Chimeric GFP-aequorin as bioluminescent Ca++ reporters at the single cell level

ABSTRACT

A modified bioluminescent system comprising a fluorescent molecule covalently linked with a photoprotein, wherein said link between the two proteins has the function to stabilize the modified bioluminescent system and allowing the transfer of the energy by Chemiluminescence Resonance Energy Transfer (CRET).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based on and claims the benefit of U.S. ProvisionalApplication Nos. 60/208,314, filed Jun. 1, 2000, 60/210,526, filed Jun.9, 2000, and 60/255,111, filed Dec. 14, 2000. The entire disclosure ofeach of these applications is relied upon and incorporated by referenceherein.

BACKGROUND OF THE INVENTION

This invention relates to a modified bioluminescent system comprising aflourescent molecule covalently linked with a photoprotein allowing thetransfer of energy by Chemiluminescence Resonance Energy Transfer(CRET). This invention also relates to the use of the modifiedbioluminescent system in in vivo and in vitro assays.

Calcium is implicated in the regulation of a great variety ofintracellular processes (1). Several techniques are most commonly usedfor intracellular Ca⁺⁺ monitoring. Patch-clamp and Ca⁺⁺ selectivemicroelectrodes give cumulative measurements of Ca⁺⁺ fluxes in arestricted number of cells. On the other hand, intracellular [Ca⁺⁺]dynamics in large populations of cells can be visualized withfluorescent probes (2). Genetic tools could provide new methods for Ca⁺⁺monitoring.

Two groups of genetic Ca⁺⁺ probes are at present available. The firstcategory uses the principle of Fluorescence Resonance Energy Transfer(FRET) between two variants of the green fluorescent protein (GFP). Thetwo GFP are covalently linked by a calmodulin binding sequence alone orin combination with calmodulin so that intramolecular FRET does (3) ordoes not (4) occur in response to Ca⁺⁺ influx. The second category iscomposed by bioluminescent proteins, such as aequorin (5, 6). The activeprotein is formed in the presence of molecular oxygen from apoaequorin(189 amino acids) and its luciferin, coelenterazine (Mr 423) (7).

The binding of Ca⁺⁺ to aequorin, which has three EF-hand structurescharacteristic of Ca⁺⁺ binding sites, induces a conformational changeresulting in the oxidation of coelenterazine via an intramolecularreaction. Moreover, the coelenteramide so produced is in an excitedstate, and blue light (max: 470 nm) is emitted when it returns to itsground state (8). Such a bioluminescent genetic marker presents theadvantage over Ca⁺⁺-sensitive fluorescent dyes of being easily targetedto specific cells and in subcellular compartments with appropriateregulatory elements and peptide signals (9). The bioluminescent processdoes not require light excitation like fluorescent probes or proteins,and thus does not induce autofluorescence, photobleaching and biologicaldegradation problems. Furthermore, aequorin is not toxic, does not bindother divalent cations and does not interfere with the [Ca⁺⁺], buffersystem even when microinjected at high concentrations. Its low affinityfor Ca⁺⁺ (Kd=10 (μM) is probably responsible for this and makes aequorina good sensor in the range of biological [Ca⁺⁺] variations.

Although providing a good ratio of signal over background, aequorinsignals are very difficult to detect because of aequorin's low lightquantum yield, that is, the number of emitted photons per protein thatbind Ca⁺⁺. In the jellyfish, Aequorea victoria, from which aequorin hasbeen isolated (10), the protein is associated with the GFP (11). AfterCa⁺⁺ binding, the energy acquired by aequorin is transferred from theactivated oxyluciferin to GFP without emission of blue light. The GFPacceptor fluorophore is excited by the oxycoelenterazine through aradiationless energy transfer. Then, a green light (max, 509 nm) isemitted when the excited GFP returns to its ground state (12).

Such intermolecular radiationless energy transfer is not unusual inbioluminescence and has already been shown to increase the quantum yieldof the bioluminescent process in Renilla, another coelenterate (13). Thegain measured in vitro ranges from 3 to 5 fold (14). It is possible toreconstitute in vitro the Renilla system and obtain the spectral shiftwith low equimolar concentrations of its components because theluciferase and the green fluorescent protein bind together (14).

In the Aequorea system, binding between purified photoprotein and GFPdoes not occur in solution, even when present at high concentrations(15). In vivo, energy transfer occurs because of the high concentrationof GFP. It can be obtained in vitro through the co-adsorption ofaequorin and GFP on DEAE cellulose membranes (15). The Förster equationshows that the efficiency of this process depends on several conditionsdescribed in the case of FRET. The emission spectrum of the donor musthave the greatest overlap with the excitation spectrum of the acceptor.The energy transferred is also strongly dependent on the geometry, inparticular, the relative orientation and distance of the two dipoles andmodulated by their respective motion (16).

An aim of this invention is to develop a dual reporter gene combiningproperties of Ca⁺⁺-sensitivity and fluorescence of aequorin and GFP,respectively. The fusion protein, which can be detected with classicalepifluorescence, can be used to monitor calcium activities. Theconfiguration of the molecules of the invention increases their overallturnover and allows an efficient intramolecular ChemiluminescenceResonance Energy Transfer (CRET). As a result, the quantum yield ofaequorin appears to be higher. This invention shows that physiologicalcalcium signals can be visualized in single eukaryotic cells with anintensified CCD camera. Other constructs described here target thefusion protein to the neurite membrane.

SUMMARY OF THE INVENTION

This invention thus provides a modified bioluminescent system comprisinga fluorescent molecule covalently linked with a photoprotein, whereinthe link between the two proteins has the function to stabilize themodified bioluminescent system and allow the transfer of the energy byChemiluminescence Resonance Energy Transfer (CRET). In a preferredembodiment, the bioluminescent system comprises an GFP proteincovalently linked to a aequorin protein, wherein the link between thetwo proteins has the function to stabilize the modified bioluminescentsystem and to allow the transfer of the energy by ChemiluminescenceResonance Energy Transfer (CRET).

In one embodiment of a modified bioluminescent system according to theinvention, the bioluminescent system comprises an GFP protein covalentlylinked to an aequorin protein, wherein the link between the two proteinsis constituted by at least 5 amino acids and optionally at least 5 aminoacids and at least one copy of 9 amino acids. The link has the functionof stabilizing the system and allowing the transfer of energy byChemiluminescence Resonance Energy Transfer (CRET).

In a preferred embodiment, the bioluminescent system comprises a GFPprotein covalently linked to an aequorin protein, wherein the linkbetween the two proteins is preferably constituted by at least 5 aminoacids and five copies of 9 amino acids and has the function ofstabilizing the system and allowing the transfer of energy byChemiluminescence Resonance Energy Transfer (CRET).

The two proteins can be separate or together functional. In addition,the modified bioluminescent system can be calcium sensitive and/or lightsensitive.

This invention also provides a method of screening in vitro a change ina physical, chemical, biochemical, or biological condition. The methodcomprises:

a) providing in different samples a bioluminescent system according tothe invention in a reaction system containing an analyte of interest;

b) measuring whether light is produced; and

c) detecting a change based on the production of light.

Further, this invention provides a method of screening in vivo a changein a physical, chemical, biochemical, or biological condition. Themethod comprises the steps of:

a) administering to a mammal an acceptable composition comprising abioluminescent system according to the invention;

b) detecting whether light is produced; and

c) optionally measuring ionic concentration of calcium flux.

In addition, this invention provides a composition comprising a purifiedpolypeptide, wherein the composition has the functional characteristicsof binding calcium ions and transmitting measurable energy, said energydepending on the quantity of calcium bound and on the quantity ofpolypeptides in said composition in absence of any light excitation.

In addition, this invention provides a purified polypeptide having theamino acid sequence of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ IDNO: 4; SEQ ID NO: 5; and SEQ ID NO: 6.

In other embodiments, this invention provides a polynucleotide havingthe sequence of SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10;SEQ ID NO: 11; and SEQ ID NO: 12.

This invention also provides a culture as deposited at the C.N.C.M. andcontaining the plasmid No. I-2507; the plasmid No. I-2508; the plasmidNo. I-2509; the plasmid No. I-2510; the plasmid No. I-2511; the plasmidNo. I-2512; or the plasmid No. I-2513.

Further, this invention provides a peptide linker having the functionafter translation to approach a donor site to an acceptor site inoptimal conditions to permit a direct transfer of energy bychemiluminescence in a purified polypeptide according to the invention.The nucleotide linker can have, for example, the nucleotide sequence ofSEQ ID No: 13; SEQ ID No: 14; SEQ ID No: 15; SEQ ID No: 16, or SEQ IDNo: 17. The peptide linker can comprise at least 5 amino acids andcomprising the amino acid sequence of SEQ ID No: 18; SEQ ID No: 19; SEQID No: 20; SEQ ID No: 21, or SEQ ID No: 22.

A kit for measuring the transfer of energy in vivo or in vitro containsat least one of the polypeptides according to the invention or thepolynucleotide according to the invention and the reagents necessary forvisualizing or detecting the said transfer in presence or in absence ofa molecule of interest.

In another embodiment, the invention provides a fusion protein of theformula:

GFP-LINKER-AEQ;

wherein GFP is green fluorescent protein; AEQ is aequorin; and LINKER isa polypeptide of 4-63 amino acids, preferably 14-50 amino acids.

The LINKER can comprise the following amino acids:

(Gly Gly Ser Gly Ser Gly Gly Gln Ser [SEQ ID NO: 25])_(n), wherein n is1-5. Preferably n is 1 or n is 5. LINKER can also include the amino acidsequence Ser Gly Leu Arg Ser [SEQ ID NO: 26].

Another fusion protein for energy transfer from aequorin to greenfluorescent protein by Chemiluminescence Resonance Energy Transfer(CRET) following activation of the aequorin in the presence of Ca⁺⁺ hasthe formula:

GFP-LINKER-AEQ;

wherein GFP is green fluorescent protein; AEQ is aequorin; and LINKERcomprises the following amino acids:

(Gly Gly Ser Gly Ser Gly Gly Gln Ser [SEQ ID NO: 25])_(n), wherein n is1-5; and wherein the fusion protein has an affinity for Ca⁺⁺ ions and ahalf-life of at least 24 hours. The LINKER can include the amino acidsequence Ser Gly Leu Arg Ser [SEQ ID NO: 26]. In addition, the fusionprotein can further comprise a peptide signal sequence for targeting thefusion protein to a cell or to a subcellular compartment.

This invention also provides polynucleotides encoding fusion proteins asdescribed above.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention will be described with reference to the drawings inwhich:

FIG. 1 is a schematic map of different constructions. All the constructswere under the control of the human cytomegalovirus promoter (PCMV). Anasterisk indicates the position of a Val-163-Ala mutation. In pGA, thecoding sequences of GFP and aequorin are separated by 5 codons. One tofive linkers (in brackets) have been added in pG_(i)A where i is thenumber of linker. Linkers were oriented so as to encode a 9 amino acidrepeat. Complete Synaptotagmin 1 or its transmembrane part (tSyn) werefused in frame with the G5A.

FIG. 2 depicts Ca⁺⁺ CRET activities on cellular extracts. Emissionspectra of aequorin and several GFP-Aequorin fusion proteins werecalibrated as a percentage of maximum intensity. CRET measurements areexpressed as the ratio of green (500 nm) over blue (450 nm) photons.

FIG. 3 depicts GFP fluorescence of GFP-Apoaequorin proteins in Neuro2Acells transfected with pGm (A), pGA (B), pG2A (C), and pG5A (D).Confocal superposition of GFP fluorescence and immunostaining ofsynaptotagmin in cells expressing either pSG5A (E) or pStG5A (F) isshown.

FIG. 4 depicts Ca⁺⁺-induced bioluminescence detected at the single celllevel. Neuro2A cells transfected with pGA (A. 1-4) or pSG5A (B) werepre-incubated with 5 μM coelenterazine in a Ca⁺⁺-free buffer. (A.3) GFPFluorescence made it possible to choose transfected cells. Thebackground recorded before CaCl₂ addition (A.2) corresponds to therelative light unit (RLU) level at time 0 of experiment (A.4, B).Intensities of fluorescence and bioluminescence activity are translatedin scaled pseudocolors. Representative pictures of the chosen field areshown after addition of 5 mM CaCl₂ and 5 μM A23187 at 13 sec. and 159sec, respectively, after the beginning of the acquisition (A.1). (A.4)Each profile indicates the intensity of light emitted by a single cell.

Five regions of interest were defined by encircling individual cellsoma. With pGA (data not shown) or pSG5A (B) transfection, a highconcentration of CaCl₂, (100 mM) was added at the end of the experiment(500 sec.) to check that the bioluminescent protein was still active.(C) Control experiments were made with Fluo-3 AM on mock-transfectedNeuro2A cells.

FIG. 5 depicts the results of analysis of protein stability for variousfusion proteins.

FIG. 6 depicts the results of the determination of the Ca⁺⁺ affinity ofaequorin and fusion protein G5A.

FIG. 7 Calibration curves between the bioluminescent activity and Ca2+.

FIG. 8 Fluorescence and Ca2+-induced bioluminescent activity indissociated neurons in culture infected with adenoviral-G5A vectors.

FIG. 9 Fluorescence and Ca2+-induced bioluminescent activities indissociated neurons in culture infected with adenoviral-SG5A vectors.

FIG. 10 shows representative pattern of luminescence activity afterinjection of GA plasmid at the one cell stage of Xenopus embryo.

FIG. 11 shows a transgenic Xenopus larva with GFP-aequorin.

DETAILED DESCRIPTION OF THE INVENTION

Among the coelenterates, bioluminescent species exist. Numerous studieshave shown that the bioluminescence is generated by photoproteins thatare sensitive to calcium. These proteins emit a flash of light inresponse to an increase in the concentration of calcium ions. Amongthese photoproteins, aequorin is one of the most well studied (Blinks etal., 1976).

Isolated in the jellyfish, Aequoria victoria (Shimomura et al., 1962),aequorin, after binding with three calcium ions, emits a flash of bluelight with a spectrum of maximum wavelength 470 nm. Contrary to aclassical luciferase-luciferin reaction, the emission of light does notrequire oxygen, and the total amount of light is proportional to theamount of protein. Oxygen is necessary, however, to reconstitute theaequorin, by the action of apoaequorin, a protein with a molecular massof 21 kDa, and coelenterazine. The emission of photons is caused by aperoxidation reaction in the coelenterazine, after binding with thethree calcium ions on the aequorin. Two hypotheses have been suggestedfor this process: (i) the binding between aequorin and calcium ionsinduces the emission of light by a conformational change in the protein,allowing oxygen to react with coelenterazine, and (ii) oxygen plays arole in the binding between coelenterazine and apoaequorin (Shimomuraand Johnson, 1978). Aequorin may be recreated in vitro and in vivo byeliminating oxyluciferin, adding luciferin (coelenterazine) in thepresence of β-mercaptoethanol and oxygen (Shimomura and Johnson, 1978).The necessity of adding β-mercaptoethanol or a reducing agent toreconstitute aequorin is presumably due to the presence of at least onesulfhydryl group of cysteine 145 included in a negatively chargedmicroenvironment (Charbonneau et al., 1985).

More than thirty semi-synthetic aequorins having different affinitiesfor calcium ions have been characterized, based on the type ofcoelenterazine that binds to the protein (Shimomura, 1991; incorporatedby reference herein). The dissociation constant between aequorin and thecalcium ions is estimated to be between 0.1 mM (Allen et al., 1997) and1 mM (Prasher et al., 1985). Although the relationship between lightemission and calcium ion concentration may not be linear, a logarithmicrelationship between the emission of light and the calcium ionconcentration has nonetheless been determined (Johnson and Shimomura,1978). Indeed, a 200-fold increase in the signal to background noiseratio is measured when the Ca⁺⁺ concentration goes from 10⁻⁷M to 10⁻⁶M,and by a factor of 1000, from 10⁻⁶M to 10⁻⁵M (Cobbold and Rink, 1987).Moreover, the kinetics of the signal emission is rapid enough to detecttransitory increases in Ca⁺⁺ ion concentrations. An increase in lightintensity with a time constant of 6 msec, under calcium saturationconditions, has been shown (Blinks et al., 1978). Aequorin is thus aphotoprotein that is well adapted to measure rapid and elevatedincreases in Ca⁺⁺ ions under physiological conditions.

The cloning of the apoaequorin gene by Prasher et al., (1985) and Inouyeet al. (1985) has led to the creation of expression vectors, makingpossible its targeting in a specific cell compartment by fusion withnuclear, cytoplasmic, mitochondrial, endoplasmic reticulum, or plasmamembrane signal peptides (Kendall et al., 1992; Di Giorgio et al.,1996). In addition, the in vivo expression of the protein makes possibleits detection at low levels, leaving the intracellular physiology ofcalcium undisturbed.

In nature, photoprotein activity is very often linked to a secondprotein. The most common is the “green flourescent protein” or GFP. Thelight emitted in this case is in fact green. The hypothesis of an energytransfer between aequorin and GFP by a radiative mechanism was proposedin the 1960s by Johnson et al., (1962). The blue light emitted byaequorin in the presence of Ca⁺⁺ is presumably absorbed by GFP andreemitted with a spectrum having a maximum wave length of 509 nm. otherstudies have shown that this transfer of energy occurs through anon-radiative mechanism made possible through the formation ofheterotetramer between GFP and aequorin. Morise et al. (1974) havesucceeded in visualizing this energy transfer in vitro, and aco-adsorption of the two molecules on a DEAE-cellulose membranefacilitates the process. Through this mechanism, it thus appearspossible to increase the quantum efficiency of the system (Ward andCormier, 1976).

GFP, also isolated in the jelly fish Aequoria victoria, was recentlycloned (Prasher et al., 1992). It has been used in different biologicalsystems as a cellular expression and lineage marker (Cubitt et al.,1995). Detecting this protein using classical fluorescence microscopy isrelatively easy to do in both living organisms and fixed tissue. Inaddition, fluorescent emission does not require the addition of acofactor or coenzyme and depends on an autocatalytic post-translationalprocess. The fluorophore, consisting of nine amino acids, ischaracterized by the formation of a cycle between serine 65 and glycine67, which gives rise to an intermediate imidazolidine 5, followed byoxidation of tyrosine 66, transforming it into dehydrotyrosine (Heim etal., 1994). This group is found inside a cylinder composed of 11 βlayers, which constitutes an environment that interacts directly withthe chromophore (Yang et al., 1996).

Monitoring calcium fluxes in real time could help to understand thedevelopment, the plasticity, and the functioning of the central nervoussystem. In jellyfish, the chemiluminescent, calcium binding, aequorinprotein is associated with the green fluorescent protein (GFP), and agreen bioluminescent signal is emitted upon Ca⁺⁺ stimulation. Aequorinalone is difficult to detect on the cellular and subcellular level owingto the weak emission of photons after excitation.

The development of a new marker sensitive to calcium with a higherquantum yield was therefore initiated. This invention utilizesChemiluminescence Resonance Energy Transfer (CRET) between the twomolecules. Calcium sensitive bioluminescent reporter genes have beenconstructed by fusing GFP and aequorin resulting in much more lightbeing emitted.

Chemiluminescent and fluorescent activities of these fusion proteinshave been assessed in mammalian cells. Cystosolic Ca⁺⁺ increases wereimaged at the single cell level with a cooled intensified CCD (coupledcharge device) camera. This bifunctional reporter gene should allow theinvestigation of calcium activities in neuronal networks and in specificsubcellular compartments in transgenic animals.

GFP-aequorin Fusion Proteins as Ca⁺⁺-Activated Reporter Genes.

According to this invention, a fusion protein has been constructed withaequorin and GFP to increase the quantum yield of Ca⁺⁺-inducedbioluminescence. This activity can not be increased simply byco-expressing GFP with aequorin (data not shown). A thermoresistant GFP(Gm) was fused in frame with the NH₂ terminal region of apoaequorin(FIG. 1), since the C-terminal proline residue has been shown to beimplicated in the Ca⁺⁺-activated bioluminescent process (20).

Different constructs have been made with increasing size of linkerbetween GFP and apoaequorin. The shortest spacer is formed by 5 aminoacids and the longest by 50 amino acids (FIG. 1). All the fusionproteins showed a better Ca⁺⁺-triggered bioluminescent activity thanaequorin alone. The increases of light emitting activity ranged from 19to 65 times (Table 1) possibly because of greater protein stability.

TABLE 1 CA++ INDUCED CHEMILUMINESCENCE ACTIVITIES Mean ± SEM* Name RLU ×10⁶/10 Uβgal pA  0.15 (0.10; 021)  pGa 10.01 ± 4.4 pG1A 2.96 (3.39;2.53) pG2A 8.39 (9.54; 7.23) pG4A  7.78 (12.02; 3.53) pG5A  8.15 ± 1.72*SEM is indicated when more than two measures were made. Otherwise thetwo measures are given.

The plasmids identified in Table 1 are described in detail hereafter.The following sequence identifiers are used to describe the amino acidand nucleotide sequences of each plasmid insert.

TABLE 2 SEQUENCE IDENTIFIERS Plasmid Insert Amino Acid SequenceNucleotide Sequence A * * GA  SEQ ID NO: 1 SEQ ID NO: 7 G1A SEQ ID NO: 2SEQ ID NO: 8 G2A SEQ ID NO: 3 SEQ ID NO: 9 G4A SEQ ID NO: 4  SEQ ID NO:10 G5A SEQ ID NO: 5  SEQ ID NO: 11 SeG5A SEQ ID NO: 6 12 * Thenucleotide sequence of apoaequorin is contained in U.S. Pat. No.5,422,266.

The identity of the linker used in these constructs is as follows:

DNA sequence of GFP-aequorin linker pGA (strain I-2507) TCC GGC CTC AGATCT [SEQ ID NO:13] pG1A (strain I-2508) TCC GGC GGG AGC GGA TCC GGC GGCCAG TCC [SEQ ID NO:14] GGC CTC AGA TCT pG2A (strain I-2509) TCC GGC GGGAGC GGA TCC GGC GGC CAG TCC [SEQ ID NO:15] GGC GGG AGC GGA TCC GGC GGCCAG TCC GGC CTC AGA TCT pG4A (strain I-2510) TCC GGC GGG AGC GGA TCC GGCGGC CAG TCC [SEQ ID NO:16] GGC GGG AGC GGA TCC GGC GGC CAG TCC GGC GGGAGC GGA TCC GGC GGC CAG TCC GGC GGG AGC GGA TCC GGC GGC CAG TCC GGC CTCAGA TCT pG5A (strain I-2511) TCC GGC GGG AGC GGA TCC GGC GGC CAG TCC[SEQ ID NO:17] GGC GGG AGC GGA TCC GGC GGC CAG TCC GGC GGG AGC GGA TCCGGC GGC CAG TCC GGC GGG AGC GGA TCC GGC GGC CAG TCC GGC GGG AGC GGA TCCGGC GGC CAG TCC GGC CTC AGA TCT pSeG5A (strain I-2512) and pStG5A(strain I-2513) same linker sequence as pG5A.

Peptide sequence of linker pGA Ser Gly Leu Arg Ser [SEQ ID NO:18] pG1ASer Gly Gly Ser Gly Ser Gly Gly Gln Ser Gly Leu Arg Ser [SEQ ID NO:19]pG2A Ser Gly Gly Ser Gly Ser Gly Gly Gln Ser Gly Gly Ser Gly [SEQ IDNO:20] Ser Gly Gly Gln Ser Gly Leu Arg Ser pG4A Ser Gly Gly Ser Gly SerGly Gly Gln Ser Gly Gly Ser Gly [SEQ ID NO:21] Ser Gly Gly Gln Ser GlyGly Ser Gly Ser Gly Gly Gln Ser Gly Gly Ser Gly Ser Gly Gly Gln Ser GlyLeu Arg Ser pG5A Ser Gly Gly Ser Gly Ser Gly Gly Gln Ser Gly Gly Ser Gly[SEQ ID NO:22] Ser Gly Gly Gln Ser Gly Gly Ser Gly Ser Gly Gly Gln SerGly Gly Ser Gly Ser Gly Gly Gln Ser Gly Gly Ser Gly Ser Gly Gly Gln SerGly Leu Arg Ser pSeG5A and pStG5A idem than pGSA

Plasmids containing the foregoing polynucleotides have been deposited atthe Collection Nationale de Cultures de Microorganismes (“C.N.C.M.”),Institut Pasteur, 28, rue du Docteur Roux, 75724 Paris Cedex 15, France,as follows:

Insert Plasmid Accession No. Deposit Date A pAeq+ I-2506 Jun. 22, 2000GA  pGa   I-2507 Jun. 22, 2000 G1A pG1A I-2508 Jun. 22, 2000 G2A pG2AI-2509 Jun. 22, 2000 G4A pG4A I-2510 Jun. 22, 2000 G5A pG5A I-2511 Jun.22, 2000 SeG5A pSeG5A I-2512 Jun. 22, 2000 StG5A pStG5A  I-2513 Jun. 22,2000

Recombinant apoaequorin is unstable within the cytosol, with a half-lifeof approximately 20 minutes (21). In contrast, GFP is a very stableprotein and probably stabilizes apoaequorin in the chimeric proteins.The turnover times of the different cytosolic proteins were estimated ontransient expression in COS 7 cells by treatment with puromycin (50μg/ml) for 6 hours. Over this period, most fusion proteins presented a30% decrease of activity compared with the 80% loss of apoaequorin whenalone (FIG. 5). It has been observed that, in vitro, the fusion proteinsof the invention were more sensitive than aequorin alone. G5A gives asignificant signal over background with Ca⁺⁺ concentration as low as 38nM, whereas aequorin needs 28 times more calcium (1 μM) to yield acomparable signal (FIG. 6). Energy transfer may also improve the quantumyield of GFP-aequorin allowing a more efficient calcium ions detection.To discriminate among the factors contributing to the higher lightemission, it will be necessary to study the relaxation mechanisms of theGFP fluorescent excited state on purified hybrid proteins.

More generally, one embodiment of this invention provides a chimericprotein starting with the genes for GFP and aequorin. Improved quantumyield will depend on the functional coupling of the proteins by aChemiluminescence Resonance Energy Transfer (CRET) mechanism. Thus,after the reconstitution of aequorin and its binding with calcium ions,the activated aequorin transmits its energy to the GFP, which in turnemits a green light to return to its ground state. Optimizing thefunctional coupling between the two proteins has focused on threepoints:

1. Improving the induction of a conformational change in the GFP at37°C., which leads to a higher emission of GFP in the mammalian cells;

2. Changing to the use of aequorin codons adapted to mammalian cells toenhance its expression; and

3. Adding a linkage peptide between the two proteins.

With respect to the third point, an initial molecular construct withfive amino acids separating the two proteins was first completed. Then asequence of nine amino acids was added in a sequence of one to fivecopies. These constructs were placed in a eukaryote expression vectorunder control of the CMV (cytomegalovirus) promoter allowing theirfunctional ability to be confirmed. These fusion proteins may beidentified: (i) by the GFP signal, through excitation of the biologicalpreparations with light of wavelength 470 nm, by fluorescence microscopy(FITC filter); (ii) by aequorin activity, through emission of blue lightafter binding with Ca⁺⁺ ions.

The following terms have the following meanings when used herein:

Luminescence

Emission of an electromagnetic radiation from an atom or molecule in UV,in visible or IR. This emission results from the transition from anelectronically excited state towards a state from weaker energy,generally the ground state.

Fluorescence

Fluorescence produced by a singlet, very short, excited electronically.This luminescence disappears at the same time as the source fromexcitation.

Chemiluminescence

Luminescence resulting from a chemical reaction.

Bioluminescence

Visible chemiluminescence, produced by living organisms. The inventionmimics the system naturally present in the jellyfish, without fixationto a support.

Bioluminescent System

The bioluminescent system according to the invention is a chimerictripartite molecule within the middle a peptide linker and a coenzyme(i.e., coelenterazine). The first molecule and the second moleculecovalently attached with the linker can be everything if they have forthe first a donor site and for the second an acceptor site attached onit (receptors-linker-ligand, antibody-linker antigen). The chimericprotein can be fused to a fragment of tetanus toxin for its retrogradeand transynaptic transport on axon by Coen, L., Osta, R., Maury, M., andBrulet, P., Construction of hybrid proteins that migrate retrogradelyand transynaptically into the central nervous system. Proc. Natl. Acad.Sci. (USA) 94 (1997) 9400-9405, or fused to a membrane receptor.

Non-Radiative

No emission of photon from aequorin to the GTP when aequorin is boundedby calcium ions (therefore there is no transmission of blue light byaequorin in the invention, the energy transfer is directly made betweenthe two proteins).

FRET System

Transfer of energy by resonance by fluorescence (i.e., between twovariants of GFP).

References

Fluorescent indicators for Ca2+ based on green fluorescent proteins andcalmodulin.

Miyawaki, A., Llopis, J., Heim, R., McCaffery, J. M., Adams, J. A.,Ikura, M. and Tsien, R. Y. Nature, (1997) Vol. 388 pp. 882-887.

Detection in living cells of Ca2+-dependent changes in the fluorescenceemission of an indicator composed of two green fluorescent proteinvariants linked by a calmodulin-binding sequence. A new class offluorescent indicators.

Romoser, V. A., Hinkle, P. M. and Persechini, A., J. Biol. Chem., (1997)Vol. 272, pp. 13270-13274.

CRET

Transfer of energy by resonance by chemiluminescence (i.e., fusionprotein with GFP-aequorin (jellyfish Aequorea) but without linker orGFP-obeline).

Reference

Chemiluminescence energy transfer.

Campbell, A. K., in Chemiluminescence: Principles and application inBiology and Medicine, Eds Ellis Horwood, Chichester, UK 1988, pp.475-534.

BRET

Transfer of energy by resonance by bioluminescence (i.e., interactionbetween GFP and luciferase jellyfish Renilla).

Reference:

A bioluminescence resonance energy transfer (BRET) system: applicationto interacting circadian clock protein.

Xu, Y., Piston, D. W. and Johnson, C. H. Proc. Natl. Acad. Sci., (USA)(1999) Vol. 96, pp. 151-156.

Application 1: Study of Calcium Signals from a Cell Population in aEukaryotic Organism.

Targeting the bioluminiscent protein sensitive to calcium in a cellpopulation or in a specific tissue may be achieved through homologousrecombination or by transgenesis under the control of a specificpromoter. Replacing genes by homologous recombination in embryonic cellsin mice, such, as Hoxc-8 and Otxl, with this new marker will make itpossible to obtain new lines of mutant mice. This approach will permitthe detection of electrical activity in a group of neural cells, andwill also make it possible to complete the phenotype study of mutantsobtained by replacing the LacZ gene (Le Mouéllic et al., 1990, 1992;Acampora et al., 1996). For the Hoxc-8 locus, the expression of themarker should be located in the ventral horns of the spinal chordbeginning at section C7 (Le Mouellic et al., 1990). Anomalies in thesomatotopic organization of the motor neurons innervating these muscleshave been brought to light (Tiret et al., 1998), and a study of the roleof the flux of calcium in the establishment of these neural connectionsduring development may thus be undertaken. In the Otxl model, thetransgene should be expressed in specific regions of the forebrain,given that an expression localized at layers V and VI of the cerebralcortex, and in regions of the diencephalon, mesencephalon, andcerebellum have been shown (Frantz et al., 1994). Mutant mice obtainedby the replacement of the gene by the LacZ gene show a reduction in thethickness of the cerebral cortex and anomalies in the hippocampus,mesencephalon, and cerebellum (Acampora et al., 1996). The loss ofbalance and rotatory movement observed in these mice can presumably beattributed to anomalies in the sensory organs, specifically in the eyeand inner ear. These mice are also subject to generalized epilepticseizures. The establishment of faulty connections and/or abnormalelectrical activity could be implicated in the genesis of thesepathological processes (McNamara, 1992). The use of this new markerwill, on the one hand, make it possible to verify these hypothesesthrough a functional and dynamic approach, and on the other, to addressthe development of epilepsy in the adult as well as during development.

Application 2: Study of the Role of Intraceilular Calcium

Calcium is involved in a large number of cellular mechanisms, such ascellular migration, membrane excitability, mitochondrial metabolism,secretion, mitosis, and synaptic plasticity (Berridge et al., 1998).Coding calcium information at the cellular and subcellular level iscomplex, involving spatial, temporal and quantitative factors. Targetingmarker of the invention to different subcellular compartments ispossible by fusion with a peptide signal, for example, synoptotagmine.

Example A: Targeting the nuclear compartment will make it possible tostudy the role of calcium in transcription activation mechanisms andduring the mechanisms related to programmed cell death (apoptosis).

Example B: Targeting two fusion proteins with GFP produces differentemission spectra in the two cell compartments, for example, cytoplasmand the endoplasmic reticulum will make it possible to study theregulation of the calcium flux during cell activations.

Example C: Targeting the fusion protein in the synapses will make itpossible to study the calcium activity linked to electrical activity inneural cells during the release of neurotransmitters. The firstpossibility is the achievement of a triple fusion between a synapticprotein, such as synaptotagmine or SNAP25, GFP, and aequorin. Theexistence of protein—protein interactions during exocytosis makes itpossible to consider a second possibility: A functional coupling betweenGFP and aequorin, the one in fusion with a vesicular protein and theother with a plasma protein. A signal will be obtained only during theinteraction of the different proteins in the presence of an increase inthe calcium ion concentration.

Application 3: Study of Calcium Signals at the Cell Population Level

Triple fusing of a protein having intercellular transport propertiessuch as fragment C of the tetanus toxin (TTC) or the VP22 protein of theherpes virus with GFP and aequorin will make it possible to observe thecalcium activity in a population of connected cells, for example in aneural network.

Description of the Construction of a Bioluminescent Marker ExpressionVector Sensitive to Calcium Ions

Stage 1: pEGFP-CldKS (KpnI-SmaI Deletion)

Double digestion of pEGF-Cl plasmid (Clontech, see figure) with KpnI andSmaI enzymes. After blunt ending the KpnI extension with “Mung bean”nuclease, the two extremities are ligated.

(SEQ ID NO: 27) 5′ GTC GAC GCT ACC GCG GGC CCG GGA TCC 3′ 3′ CAG CTGCCA TGG CGC CCG GGC CCT AGG 5′                KpnI            SmaI                       GTC GAC GGT AC          G   GGA TCC    CAG CTGC               C   CCT AGG                        GTC GACG               G   GGA TCC    CAG CTG C               C   CCT AGG                    (SEQ ID NO: 28)            GTC GAC GGG GAT CC           CAG CTG CCC CTA GC              SalI         BamHI

Stage 2: pEGFP-CImut (GFP Mutagenesis)

Four mutagenesis oligonucleotides were used on a single-strandedmolecule prepared using pEGFP-CIdKS. Each oligonucleotide comprises oneor several mismatches (identified below in lower case letters), causingthe desired mutation. In the pEGFP-CImut plasmid chosen, cut with theSacII enzyme but not the AgeI enzyme, all of the mutations were verifiedby sequencing.

Destruction of the AgeI site, introduction of a SacII site and deletionof a Valine codon normally absent in “wild-type” GFP (Prasher, D. C.,Eckenrode, R. K., Ward, W. W., Prendergast, F. G., and Cormier, M. J.,Primary structure of the Aequorea victoria green-flourescent protein.Gene 111 (1992) 229-233.)

(SEQ ID NO: 30) Met     Ser Lys Gly Asp (SEQ ID NO: 29)           SacIIoGM1: 5-′ GCGCTACCGcggGCCACC ATG     AGC AAG GGC GAG 3′ (SEQ ID NO: 31)pEGFP-CIdKS: 5′ GCGCTACCGGTCGCCACC  ATG GTG AGC AAG GGC GAG 3′          AgeI             Val

Replacement of the 163 Valine codon by an Alanine codon in order toincrease the quantity of GFP assuming a correct conformation at 37° C.(Siemering, K. R., Golbik, R., Sever, R., and Haseloff, J., Mutationsthat suppress the thermosensitivity-of green flourescent protein.Current Biol. 6(1996) 1653-1663.)

(SEQ ID NO: 34)       Ile Lys Ala Asn Phe Lys (SEQ ID NO: 33) oGM2:5′ GC ATC AAG Gcc AAC TTC AAG 3′ (SEQ ID NO: 35) pEGFP-CIdKS 5′ GC ATCAAG GTG AAG TTC AAG 3′ (SEQ ID NO: 36)           Val

Replacement of a 231 Leu codon by a Histidine codon normally present in“wild-type” GFP (Prasher, D. C., Eckenrode, V. K., Ward, W. W.,Prendergast, F. G., and Cormier, M. J., Primary structure of theAequorea victoria green-fluorescent protein. Gene 111(1992) 229-233.)

(SEQ ID NO: 38)       Ile Thr His Gly Met (SEQ ID NO: 37) oGM3: 5′ GGATC ACT CaC GGC ATG GA 3′ (SEQ ID NO: 39) pEGFP-CIdKS: 5′ GG ATC ACT CTCGGC ATG GA 3′ (SEQ ID NO: 38)       Ile Thr His Gly Met (SEQ ID NO: 37)oGM3: 5′ GG ATC ACT CaC GGC ATG GA 3′ (SEQ ID NO: 39) pEGFP-CIdKS: 5′ GGATC ACT CTC GGC ATG GA 3′ (SEQ ID NO: 40)               Leu

Stage 3: pEGFPmut-Aeq (GFP-Aeguorin Fusion Protein)

Four PCRs (Polymerase Chain Reaction) done on a vector comprising theaequorin (Aeq) coding phase makes it possible to amplify the A, B, C,and D fragments with, respectively, the primers oAE5A and oAE3A, oAE5Band oAE3B, oAE5C and oAE3C, oAE5D and oAE3D. The overlapping regions areused to assemble the different parts during successive PCRs (Ho, S. N.,Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R.Site-directed mutagenesis by overlap extension using the polymerasechain reaction Gene 77 (1989) 51-49.) An A+B fragment is amplifiedstarting with a mixture of A and B fragments, and primers oAE5A andoAE3B. Similarly, a C+D fragment is amplified with a mixture of C and Dfragments, using the primers oAE5A and oAE3D. Finally, the completecoding phase, A+B+C+D is developed with the primers oAE5A and oAE3D.

Each oligonucleotide comprises one or several mismatches that areidentified below in lower case. The “wild” sequence is representedopposite, in upper case. The primer oAE5A suppresses the originalinitiation translation code (ATG) and introduces a BglII site. Theprimer oAE3D introduces an XhoI site just behind the translationterminal codon (TAA). The final PCR product, digested with the BglII andXhoI enzymes, is cloned in the BglTI-SalI sites of the pEGFP-CImutplasmid in such a way that the Valine codon (GTC), the first codon ofaequorin, is the same reading phase as the GFP (see figure). The otherprimers introduce “silent” mutations that do not change the proteinsequence but modify six codons in the jellyfish, Aequoria Victoria, toimprove their expression in mammals (Wada, K-N., Aota, S.-I., Tshuchiya,R., Ishibashi, F., Gojobori, T., and Ikemura, T. Codon usage tabulatedfrom the GenBank genetic sequence data. Nucleic Acids Res. 18 suppl.(1990) 2367-2411). The completeness of the entire sequence was verifiedby sequencing,

(SEQ ID NO: 41)    oAE5A  CCATG                   5′    AGCTTCAgatct GTCAAA CTT ACA TCA GAC TTC GAG AAC CCA AGA TGG ATT GGA CGA3′    TCGAAGTctaca CAG TTT GAA TGT AGT CTG AAG CTG TTG GGT TCT ACC TAACCT GCT         BGlII CAC AAG CAT ATG TTC AAT TTC CTT GAT GTC AAC CACAAT GGA AAA ATC TCT CTT GAC GAG GTG TTC GTA TAC AAG TTA AAG GAA CTA CAGTTG GTG TTA CCT TTT TAG AGA GAA CTG CTC ATG GTC TAC AAG GCA TCT GAT ATTGTC ATC AAT AAC CTT GGA GCA ACA CCT GAG CAA GCC TAC CAG ATG TTC CGT AGACTA TAA CAG TAG TTA TTG GAA CCT CGT TGT GGA CTC GTT CGG             oAE5B  A                 AAA CGA CAC AAA GAT GCT GTg GAAGCC TTC TTC GGA GGA GCT GGA ATG AAA TAT GGT GTG TTT GCT GTGTTT CTA CGA CAc CTT CGG AAG AAG CCT CCT CGA CCT TAC TTT ATA CCA CAC                           T        oAE3A GAA ACT GAT TGG CCT GCA TATATT GAA GGA TGG AAA AAA TTG GCT ACT GAT GAA TTG GAG CTT TGA CTA ACC GGACGT ATA TAA CTT CCT ACC TTT TTT AAC CGA TGA CTA CTT AAC CTC                      oAE5C   G      T    A AAA TAC GCC AAA AAC GAA CCAACc CTC ATC CGc ATc TGG GGT GAT GCT TTG TTT GAT ATC TTT ATG CGG TTT TTGCTT GGT TGg GAG TAG GCg TAg ACC CCA CTA CGA AAC AAA CTA TAG                              C       A   T oAE3B GTT GAC AAA GAT CAAAAT GGA GCT ATT ACA CTG GAT GAA TGG AAA GCA TAC ACC AAA GCT CAA CTG TTTCTA GTT TTA CCT CGA TAA TGT GAC CTA CTT ACC TTT CGT ATG TGG TTT CGA GCTGGT ATC ATC CAA TCA TCA GAA GAT TCC GAG GAA ACA TTC AGA GTG TGC GAT ATTGAT CGA CCA TAG TAG GTT AGT AGT CTT CTA ACG CTC CTT TGT AAG TCT CAC ACGCTA TAA CTA                                            oAE5D    A      TA            GAA AGT GGA CAA CTC GAT GTT GAT GAG ATG ACA ACA CAg CAT cTgGGA TTT TGG TAC ACC CTT TCA CCT GTT GAG CTA CAA CTA CTC TAC TGT TCT GTcGTA gAc CCT AAA ACC ATG TGG                                                   T       AT       oAE3C                                                                  XhoIATG GAT CCT GCT TGC GAA AAG CTC TAC GGT GGA GCT GTC CCC TAATCTcGAGGATCTTT 3′ TAC CTA GGA CGA ACG CTT TTC GAG ATG CCA CCT CGA CAGGGG ATT AGAgCTCCTAGAAA 5′                                                                  T      oAE3D

Stage 4: pGCA (Insertion of an Intercalated Sequence)

In the pEGFPmur-Aeq plasmid, a sequence of five amino acids existsbetween the coding phases of the GFP and aequorin. Observations led tothe lengthening of this region by intercalating a sequence in the BspELsite. Two complementary oligonucleotides coding for a sequence of nineamino acids give the composition a good deal of flexibility, owing tothe abundance of Glycine and Serine. After insertion, the BspEI site ispreserved on only one side although new intercalated sequences may beadded successively. At each stage, the orientation is controlled by theBspEI enzyme. Two copies of this sequence are needed to restore thenormal fluorescence of GFP, but the energy transfer between aequorin andGFP is optimal with five copies. The entire intercalated sequence ofpGCA plasmid (5×9 aa+the five initial amino acids=50 aa) was verified bysequencing:

              Lys   Ser   Gly   Leu   Arg   Ser   Val (SEQ ID NO: 43)        5′    AAG   TCC   GGA   CTC   AGA   TCT   GTC   3′ (SEQ ID NO:42)         3′    TTC   AGG   CCT   GAG   TCT   AGA   CAG   5′ (SEQ IDNO: 44)               GFP   BspEI              BG1II      Aeq                                      5′    AAG   T     GC  GGA     CTC   AGA   TCT   GTC    3′ (SEQID NO: 45)        3′    TTC   AGG   CC    T     GAG   TCT   AGA   CAG    5′ (SEQID NO: 44)                                     +        Gly   Gly   Ser   Gly   Ser   Gly   Gly   Gln   Ser (SEQ ID NO:47) 5′  CC  GGC   GGG   AGC   GGA   TCC   GGC   GGC   CAG   T      3′(SEQ ID NO: 46)3′        G   CCC   TCG   CCT   AGG   CCG   CCG   GTC   AGG   CC   5′(SEQ ID NO: 48)                      BamHI                        BspEI

Optimization of the energy transfer by inserting a spacer between GFPand Apoaequorin.

A non-radiative energy transfer between the excited oxyluciferin and theGFP chromophore will be strongly dependent upon their overall geometryand their respective motions. Therefore, a linker was designedprincipally composed of serine and glycine residues to intercalate aflexible element of variable length.

The ratio of green and blue photons emitted upon Ca⁺⁺ triggering hasbeen measured on cellular extracts prepared 48 h after transienttransfection of Neuro2A cells. The photons emitted through abeam-splitter were counted after passing appropriate filters. Covalentlinking of GFP to aequorin (GA) significantly modified the wavelength ofmaximum light emission (FIG. 2), thereby demonstrating intramolecularenergy transfer. The ratio of green over blue light (500/450 nm) wasfurther raised from 3 to around 7 by adding 1 to 5 linkers (FIG. 2,CRET). Preliminary measurement indicates that this ratio can reachalmost 11 with SG5A probably because of the accumulation of the fusionprotein anchored to the membranes (see materials and methods).

Spectral emissions of the different constructs were also analyzed usinga monochromator. Aequorin showed a broad spectrum with maximumwavelength at 474±6.9 nm and a bandwidth, corresponding to the distancebetween low and high wavelengths at 50% values of the maximum emission,at 108.3±20.1 nm (FIG. 2). There was a clear shift toward the green inthe peak emission of the GFP-aequorin constructions ranging from506.7±1.2 nm to 514.1±3.4 nm. Increasing the length of the linkerfurther affected the sharpness of the spectrum, as indicated by thenarrower bandwidths, 88.4+9.4 nm and 56.0±3.3 nm, for pGA and pG5Arespectively. There was no evidence of a bimodal spectrum with any ofthe pG1A-pG5A constructs indicating an optimal transfer which could beincomplete in the case of pGA.

When the spacer between GFP and aequorin is longer than 14 amino acids,the donor and the acceptor dipoles have probably more freedom to be in aconfiguration favourable for optimum intramolecular energy transfer. Thesystem of the invention yields an efficiency comparable to theintermolecular CRET measured in vivo (22, 23) and provides a convenientmodel for the biophysical studies of radiationless energy transfermechanisms.

Cellular localization and targeting of GFP-Apoaequorin.

The cellular localization of the GFP-apoaequorin constructs has beenexamined. FIG. 3 illustrates GFP activity 48 h after transienttransfection in Neuro2A cells. Expression of the mutant GFP alone (Gm)showed homogenous fluorescence in the cytosol as well as in the nucleusas expected since GFP is a small protein that can diffuse into thenucleus. Mutation VI63A improves remarkably the fluorescence signal andreduces photobleaching when compared to the original EGFP (data notshown) probably owing to a higher concentration of properly foldedprotein. An evenly distribution is also observed for all theGFP-apoaequorin constructions in Neuro2A cells (FIGS. 3A-D) as well asin COS-7 cells. Bright spots often appeared in the cytosol with fusionproteins having the shortest linkers: GA, G1A and G2A. These spots wereless frequent with G4A and never observed with Gm and G5A. Highconcentrations of proteins expressed during transient transfectionscould induce the aggregation of GFP (24), which is also going to beinfluenced by the presence of the aequorin protein and the distanceseparating them.

The GFP-apoaequorin has also been targeted to the neurotransmittervesicles with a complete or a partial synaptotagmin I molecule.Synaptotagmin I is a transmembrane protein of synaptic vesicles and isimplicated in neurotransmitter exocytosis (25). For imaging calciummicrodomains in presynaptic compartments, the signal should be moreaccurate than if evenly distributed in the cytoplasm of neurons. In athree part fusion protein, SG5A (FIG. 1), the complete coding sequenceof synaptotagmin I has been put in frame upstream of G5A. In this case,GFP fluorescence is superimposable with synaptotagmin immunostaining butis also visible at the cellular surface (FIG. 3E). In neurons (26) andin Neuro2A cells, synaptotagmin I is localized in neuronal processes,but is undetectable in plasma membranes, probably because the dynamicmechanisms of exocytosis are followed by rapid endocytosis. WhenGFP-apoaequorin is fused with only the N-terminal part of synaptotagminincluding the transmembrane domain but lacking the cytoplasmic domain(tSG5A, FIG. 1), a strong fluorescence is restricted to the cytosol(FIG. 3F). The punctate labeling suggests that this protein is lockedinto the trans-golgi system. The correct targeting of the three partfusion molecule of the invention does not occur with tSG5A and appearsto be slowed down in the case of SG5A. When fused to the completesynaptotagmin protein, the bioluminescent marker is held back in theplasma membrane, but nevertheless labels all neurite outgrowths presentin Neuro2A cells.

Ca⁺⁺ detection in single cells.

Neuro2A cells were transiently transfected with pA, pGA, pG2A, pG5A orcotransfected with pA and pGm (FIG. 1). After aequorin reconstitutionwith native coelenterazine in Ca⁺⁺-free buffer, an emission of photonshas been measured with a classical intensified CCD camera upon theaddition of CaCl2 solution (5 mM) (FIGS. 4A.1 and 4A.4). With thenegligible background (FIG. 4A.2), integration time of 1 second isenough to record the signal in single cells (FIG. 4A.1) expressing anyof the fusion proteins. No signal could be visualized with aequorinalone or with co-expressed free GFP (data not shown). The presence ofunbound GFP does not improve aequorin chemiluminescence as we observedin vitro. Because of the low level of light produced, aequorin expressedin situ has never been detected in single cells except when targeted inmitochondria. With a cooled intensified CCD camera, Rutter et al. (1996)(27) have succeeded in detecting intramitochondrial Ca⁺⁺ signals whenaequorin is fused to cytochrome c oxidate. Transgenes encodingcytoplasmic aequorin can report calcium activities in monolayers ofcells only when photomultipliers (PMT) are used, which are moresensitive but lack the spatial resolution for single cell analysis. Thestability of GFP-aequorin fusions of the invention and the improvedlight emission have made it possible to detect physiological Ca⁺⁺signals at the level of single cells.

Calcium deficiency prior to measurements or the transfection conditionsused may induce cellular depolarization, such that opening of thevoltage dependent Ca⁺⁺ channels is likely to be responsible for the fastbioluminescent response to CaCl₂, addition (FIG. 4A). Light emissionwould then return to background level because of the desensitization ofCa⁺⁺ channels and the membrane depolarization by Ca⁺⁺-dependent K′channels (28). Fluo-3 showed a similar profile in mock transfections ofNeuro2A cells (FIG. 4C). Subsequent addition of a Ca⁺⁺ ionophore(A23187) induced a second emission of photons with comparable intensitybut with different kinetics. A lower light intensity is detectable inNeuro2A cells transfected with pSG5A (FIG. 4B). When a fluorescentcalcium probe is anchored to the inner surface of the membrane, theresponse kinetics are much quicker than when the probe is not targeted(29). The use of the bioluminescent reporter SG5A probably requires asystem with higher spatial and temporal resolutions. In any case, theresponses observed are not due to the complete consumption of aequorinas more bioluminescence can still be observed when a concentrated Ca⁺⁺solution (100 mM) is applied to cells (see FIG. 4B for example). Foreach construction, measurements have been repeated at least 4 times. Avariability of individual cells responses was observed, probably due tocell population heterogeneity. Further investigations are required tocalibrate relative light unit (RLU) versus Ca⁺⁺ concentrations.Patch-clamp techniques will also allow the identification of the type ofcalcium channels implicated in these responses and the effect ofcellular transfection on membrane potential.

The transgenes of the invention should permit imaging of electricalactivity in neural networks in whole animals. In vitro, two approacheswere used until recently. The first method is based on the coupling ofexocytosis to emission of light from synaptolucins in nerve cells (30).Light emission occurs when the luciferase, targeted inside the synapticvesicles, reacts with ATP in the extracellular space. With this system,the authors obtain signals correlated with the neurotransmitter releasebut the low light level requires very long acquisition times (over 30sec). In the second approach, fluorescence Ca⁺⁺ sensitive markers havebeen used for measurements of intracellular [Ca⁺⁺] by FRET (3, 4, 31).For single cell detection, this technique requires a sufficientconcentration of probe to discriminate the signal from the backgroundwhich is generated by autofluorescence of biological compounds and thepossibility of calcium-independent energy transfer between the two GFPs.The integration times are also relatively long, between 4 and 20seconds.

This invention thus provides new bifunctional hybrids in whichexpression patterns can be followed by GFP fluorescence while theaequorin moiety is the reporter of Ca⁺⁺ activity. Furthermore, thefunctional coupling of the two components, which follows the CRETprinciple, results in a higher amount of light emission and a greaterCa⁺⁺ sensitivity. Bioluminescent activities of these genetic markershave been assessed in single cells with a cooled intensified CCD camerain 1 second integration times. The recent development of low level lightdetection systems should allow detection of CRET signals with muchshorter integration times and higher spatial resolution. Intracellularand intercellular Ca⁺⁺ signaling can be approached in vivo in transgenicanimals in which the GFP-aequorin is targeted to a particular cellpopulation and/or to specific subcellular compartments. Particularly,calcium oscillations can then be imaged simultaneously in cells of anintegrated neural circuitry in real time.

This invention will be described in greater detail in the followingExamples.

EXAMPLE 1 Construction of GFP-Aequorin Fusion Proteins

All the constructs were made in the pEGFP-Cl vector (Clontech). The EGFPgene is codon-optimized for maximal expression in mammalian cells. Italso contains 2 mutations in the chromophore, F64L and S65T, whichmodify the excitation spectra and enhance fluorescence intensity (17).We further substituted valine 163 of the EGFP by alanine, using singlestrand mutagenesis, to improve the proper folding of the protein andincrease the fluorescence at 371C (18, 19). The aequorin codingsequence, a generous gift by M.-T. Nicolas, has been fused in frame atthe 3′ end of the EGFP gene in the BgIII/SaII sites of pEGFP-C1. Sevencodons were modified for a better expression in mammalian cells by meansof site-directed mutagenesis using PCR (polymerase chain reaction) withoverlap extension. Then, complementary oligonucleotides,5′-CCGGCGGGAGCGGATCCGGCGGCCAGT-3′ [SEQ ID NO: 23] and5′-CCGGACTGGCCGCCGGATCCGCTCCCG-3′ [SEQ ID NO: 24] were inserted at theBspEI site in the 15 bp sequence between GFP and aequorin. Conservationof the BspEI site at only one end allowed sequential addition of one tofive linker sequences (pG1A-pG5A).

Two additional fusion constructs were made in pG5A with a synapticprotein, synaptotagmin I of which the cDNA plasmid was generously giftby M. Fukuda. Sequences encoding for either the entire open readingframe or the first 134 N-terminal amino acids, comprising thetransmembrane domain of the protein, were fused in frame at the 51 endof the GFP-aequorin gene.

EXAMPLE 2 Cell Culture and Transfection

Neuroblastoma cells (Neuro2A, mouse) were grown in Dulbecco's Eaglemedium (Life Technologies—Gibco, UK) supplemented with 10% (V/V)heat-treated foetal calf serum, 2 mm glutamine (Life Technologies—Gibco,UK) and 100 units streptomycin-penicillin (Life Technologies—Gibco, UK).The cultures were incubated at 37° C. in a humidified atmospherecontaining 8 t CO₂ and transiently transected using either the CaPO₄technique or the FuGENE 6™ transfection reagent (ROCHE).

EXAMPLE 3 In Vitro Ca⁺⁺ Sensitive Chemiluminescence and CRET Activities

Cells were harvested 48 h after transfection in 250 μl of 10 mMβ-mercaptoethanol, 4 mM EDTA, 5 μM coelenterazine in PBS at 4° C. during2 to 4 hours. Cells were rinsed in 1 mM EDTA in PBS and harvested in 400μl of hypo-osmotic buffer (20 mM Tris-HCl pH 7.5/5 mM EDTA/5 mMβ-mercaptoethanol with a protease inhibitor cocktail according to themanufacturer, ROCHE), for 30 min. to 1 h at 4° C. The cell membraneswere broken by passing through a 30 gauge needle and the cellularextract was obtained after microcentrifugation at 13000 rpm for 1 h at40° C. The supernatant was harvested for all constructions but SG5A forwhich the membrane pellet was further resuspended. Calcium sensitivitychemiluminescent activity was measured in a luminometer (LUMAT LB95501E&EG BERTHOLD). Aliquots (10 μl) were placed in sample tube (with 90 μlof 10 mM Tris-HCl pH 7.5) in the luminometer and the light intensityexpressed in relative light unit (R.L.U.) was measured after theinjection of 100 μl of 50 mM CaCl₂/10 mM Tris-HCl pH 7.5 solution.

For CRET measurements, aliquots of extracts from transfected cells wereplaced in a reservoir chamber and brought into contact with an opticfibre bundle attached to a photon counting camera (PHOTEKthree-microchannel plate intensified CCD camera: PHOTEK 216). Beforecapture of signals, light passes through a monochromator allowing thespectral analysis of emitted photons. The acquisition begins 20 secondsbefore injection of CaCl₂ and carries on during 40 seconds afterinjection of the CaCl₂ solution (50 mM). For green/blue photons ratiodeterminations, the same procedure was followed but in this case thesystem measures the light emitted through blue (450 nm) and green (500nm) filters after a beam splitter.

EXAMPLE 4 GFP Fluorescence and Immunolocalization

Neuro2A cells were fixed 48 h after transfection in 4% paraformaldehydein PBS pH 7.4, rinsed in PBS, and mounted. GFP fluorescence isvisualized under a confocal Laser Scanning microscope (Zeiss,Heidelberg, Germany) which uses an argon-krypton laser operating inmulti-line mode or an Axiophot microscope with an epiluminescent system(Zeiss, Heidelberg, Germany). For immunolocalisation of the targetedGFP-aequorin, fixed cells were pre-treated with 50 mM NH₄Cl in PBS pH7.4 for 5 min. at room temperature, permeabilised in 2% BSA/0.02%Triton/goat serum solution in PBS during 1 h. Antibodies againstsynaptotagmin (StressGen SYA-130) were then applied during 2-4 hrs.Cells were then rinsed in PBS and incubated in 2% BSA/0.02% Triton inPBS with secondary antibody diluted at {fraction (1/100)} (TRITCconjugated antibody). Cells were then washed in PBS and mounted.

EXAMPLE 5 Single Cells Bioluminescence Detection

Forty-eight hours after transfection, cells were rinsed in 124 mM NaCl/5mM KCl/15 mM Hepes pH 7.4/5 mM NaHCO₃/1 mM NaH₂PO₄/0.5 mM MgSO₄/1. 5 mMCaCl₂/5.5 mM Glucose and later incubated in the same buffer withoutCaCl₂ with 5 μM coelenterazine to reconstituted aequorin, for 2 to 4 hat 37° C. and then rinsed. Calcium signals were visualized with amodified Olympus upright microscope (BHS) fitted with an BH2-RFCAepifluorescence unit recorded through a plan ×40 Olympus long workingdistance water-immersion lens (N.A. 0.7). GFP Fluorescence allowed tochoose the recording area on transfected cells. The excitation lamp wasshut off and the gain of the camera increased. Images were integratedevery second with a cooled Photonic Science extended ISIS video camera.Each profile in FIG. 4 represents the amount of light emitted over thearea that we defined around the soma of individual cells using the AxonImaging Workbench 2214 software. Intensities of fluorescence and CRETactivity are translated in scaled pseudocolors. Controls were made withFluo-3 AM on mock-transfected Neuro2A cells to check the experimentalconditions.

EXAMPLE 6 Protein Stability

The turnover times of the different cytosolic proteins were estimated ontransient expression in COS7 cells by treatment with puromycin (50μg/ml) for 6 h. Ca²⁺-induced chemiluminescence activities were performedon cellular extract obtained after the reconstitution of aequorin inpresence of 5 μM coelenterazine. Calcium sensitivity chemiluminescenceactivity was measured in a luminometer (LUMAT LB95501 E&EG BERTHOLD).Aliquots (10 μl) were placed in a sample tube (with 90 μl of 10 mMTris-HCl, pH 7.5) in the luminometer and the light intensity expressed,in relative light units (RLUs), was measured after the injection of 100μl of 50 mM CaCl₂/10 mM Tris-Hcl pH 7.5 solution. Relativechemiluminescence activities are expressed as a percentage of theactivity at the time zero (100%). The results are shown in FIG. 5. Asseen in FIG. 5, over this period, most fusion proteins presented 30%decrease of activity compared with the 80% loss of apoaequorin whenalone.

EXAMPLE 7 Determination of the Ca⁺⁺ Affinity of Aequorin and G5A

Ca²⁺ induced chemiluminescence activities were performed on cellularextract obtained after the reconstitution of aequorin in presence of 5μM coelenterazine. Calcium sensitivity chemiluminescence activity wasmeasured in a luminometer (LUMAT LP95501 E&EG BERTHOLD). Aliquots (10μl) were placed in a sample tube (with 90 μl of 10 mM Tric-HCl, pH 7.5)in the luminometer and the light intensity expressed, in relative lightunits (RLUs), was measured after the injection of 100 μl of differentCa/EGTA solutions. The results are shown in FIG. 6. As seen in FIG. 6,G5A gives a significant signal over background with Ca²⁺ concentrationsas low as 38 nM, whereas aequorin needs 28 times more calcium (1M) toyield a comparable signal.

For Chimeric GFP-Aequorin as Bioluminescent Ca²⁺ Reporters at the SingleCell Level

Concerning the invention of chimeric GFP-aequorin calcium sensitivebioluminescent reporters, we have developed new applications and we havesome preliminary data about sensitivity of GFP-aequorin proteins to Ca²⁺ions.

EXAMPLE 8 Ca²⁺ Sensitivity of G5A and SG5A: Calibration Curves BetweenBioluminescent Signals and Ca²⁺ Concentrations

Measurements of Ca²⁺ sensitivity of two constructs G5A and SG5A wereperformed on cellular extracts obtained after the reconstitution ofaequorin in presence of 5 μM colenterazine. Calcium chemiluminescenceactivity was measured in a luminometer (LUMAT LB95501 E&EG BERTHOLD).Aliquots (10 μl) were placed in a sample tube with 90 μl of 10 mMTris-HCl pH 7.5 in the luminometer and the light intensity expressed, inrelative light units (RLUs), was measured after the injection of 100 mlof different Ca/EGTA solutions (Molecular Probes Calcium CalibrationBuffer Kit). FIG. 7 shows the Ca²⁺ response curve of G5A, SG5A andaequorin. The curves represent the relationship between the ratio L/Lmaxand [Ca2+]. L is the rate of RLUs at any given [Ca2+] and Lmax is therate of RLUs at saturating [Ca2+]. These results show a much higheraffinity for Ca²⁺ of the various forms of GFP-aequorin than aequorin.

EXAMPLE 9 New Applications of GFP-Aequorin Reporters

Adenoviral vectors with GFP-aequorin were developed. Using these newconstructs, dissociated neurons from rat spinal cord in culture can betransfected with higher efficiency. FIGS. 8 and 9 depict Ca²⁺-inducedbioluminescent signals detected at the single cell level in dissociatedneuronal cells. Neuronal cells infected by adenoviral vectors with G5A(FIG. 8) or SG5A (FIG. 9) were pre-incubated with 5 μM coelenterazine ina Ca²⁺-free buffer. Intensities of fluorescence and bioluminescenceactivity are translated in pseudocolors. Representative pictures of thechosen fields are shown after the addition of 5 mM and 2.5 mM of CacCl₂,respectively, for FIGS. 8a-c & 9 a at 12 and 9 seconds. FIGS. 8d-e and 9b were obtained after addition of ionomycin and high concentration ofCaCl₂ (100 mM).

EXAMPLE 10 Expression of GFP-Aequorin Reporters in vivo in XenopusEmbryos and Measurement of Calcium Activities

Calcium signaling during early and late embryogenesis in Xenopus wasstudied. FIG. 10 shows representative pattern of luminescence activityillustrating the changes in intracellular calcium during the neuralinduction after the injection of the GA plasmid at the one cell stage inXenopus embryo. FIG. 11 shows a transgenic Xenopus larva withGFP-aequorin. These techniques can also be employed with zebrafish andmouse transgenics. These results show that these calcium reporters canbe used in a grate variety of organisms or tissues to visualize calciumactivity and to measure calcium concentrations.

In summary, the new linker useful for energy transfer by CRET system ina bioluminescent system has the following properties:

Forms:

Different amino acid sequences and peptide sequences of the linker aredescribed. Its length comprises a minimal size of 4 to 9 amino acids,which can be extended by a group of 7 to 12 amino acids (in a preferredembodiment 9 amino acids). The said group is extendable to 63 aminoacids, i.e., 9×6 times. The experiment was done, for example, with apeptide linker comprising 5 amino acids followed by 1 to 5 times of 9amino acids.

Functions:

Its first function is to approach donor sites and acceptor sites of twomolecules for a direct transmission of energy. This linker confers anoptimal environment for energy transmission by CRET.

The second function is the stabilization of the described system byincreasing the half life of aequorin because of the fusion of GFP. Theaequorin is linked to the GFP, which has a half life of more than 24hours.

Applications:

In a bioluminescent system, aptitude for protein—protein interaction.

Application of the bioluminescent system with the linker:epileptogenesis, SNC disease (visualization of the neuronal cellactivities during development and in the adult), neuromuscularconnection with the implication of homeogene HOX-C8 in the spinal cord.

Application in apoptosis with a chimeric protein comprising the linkeraccording to the invention by the visualization of the modifications ofthe intracellular calcium pools.

Visualization and precision of the role of calcium waves in livingorgans like the spleen (intra and intercellular calcium waves).

Results:

Chimeric protein is more stable by augmentation of the half-life of themolecule. Augmentation of the sensitivity for calcium ions is important.

The linker of the invention has surprising properties. The sensitivityof calcium ions of the chimeric molecule containing the aequorin and thelinker is different from that for aequorin alone. The invention providesa better sensitivity.

This linker makes it possible to attach together an aequorin moleculewith a GFP. The following reference demonstrates that the both moleculesdo not interact together without a linker: Morise, H. Shimomura, O.,Johonson, F. H. and Winant, J. (1974) Intermolecular Energy Transfer inthe bioluminescent system of Aequoria. Biochemistry 13, 2656-2662.

It is the first time that one can obtain visualization of aequorinsignal in a live single cell system (or in an alive animal).

In summary, monitoring calcium fluxes in real time could help tounderstand the development, the plasticity and the functioning of thecentral nervous system. In jellyfish, the chemiluminescent calciumbinding aequorin protein is associated with the green fluorescentprotein (GFP) and a green bioluminescent signal is emitted upon Ca⁺⁺stimulation. We decided to use this Chemiluminescence Resonance EnergyTransfer (CRET) between the two molecules. Calcium sensitivebioluminescent reporter genes have been constructed by fusing GFP andaequorin resulting in much more light being emitted. Chemiluminescentand fluorescent activities of these fusion proteins have been assessedin mammalian cells. Cystosolic Ca⁺⁺ increases were imaged at the singlecell level with a cooled intensified CCD camera. This bifunctionalreporter gene should allow the investigation of calcium activities inneuronal networks and in specific subcellular compartments in transgenicanimals.

Following are sequences and the corresponding sequence identifiersreferred to herein:

Peptide sequences:

GA M S K G E E L F T G V V P I L V E L D G D V N G H K F S V S G E G E GD A T [SEQ ID NO: 1] Y G K L T L K F I C T T G K L P V P W P T L V T T LT Y G V Q C F S R Y P D H M K Q H D F F K S A M P E G Y V Q E R T I F FK D D G N Y K T R A E V K F E G D T L V N R I E L K G I D F K E D G N IL G H K L E Y N Y N S H N V Y I M A D K Q K N G I K A N F K I R H N I ED G S V Q L A D H Y Q Q N T P I G D G P V L L P D N H Y L S T Q S A L SK D P N E K R D H M V L L E F V T A A G I T H G M D E L Y K S G L R S VK L T S D F D N P R W I G R H K H M F N F L D V N H N G K I S L D E M VY K A S D I V I N N L G A T P E Q A K R H K D A V E k F F G G A G M K YG V E T D W P A Y I E G W K K L A T D E L E K Y A K N E P T L I R I W GD A L F D I V D K D Q N G A I T L D E W K A Y T K A A G I I Q S S E D CE E T F R V C D I D E S G Q L D V D E M T R Q H L G F W Y T M D P A C EK L Y G G A V P G1A M S K G E E L F T G V V P I L V E L D G D V N G H KF S V S G E G E G D A T [SEQ ID NO: 2] Y G K L T L K F I C T T G K L P VP W P T L V T T L T Y G V Q C F S R Y P D H M K Q H D F F K S A M P E GY V Q E R T I F F K D D G N Y K T R A E V K F E G D T L V N R I E L K GI D F K E D G N I L G H K L E Y N Y N S H N V Y I M A D K Q K N G I K AN F K I R H N I E D G S V Q L A D H Y Q Q N T P I G D G P V L L P D N HY L S T Q S A L S K D P N E K R D H M V L L E F V T A A G I T H G M D EL Y K S G G S G S G G Q S G L R S V K L T S D F D N P R W I G R H K H MF N F L D V N H N G K I S L D E M V Y K A S D I V I N N L G A T P E Q AK R H K D A V E A F F G G A G M K Y G V E T D W P A Y I E G W K K L A TD E L E K Y A K N E P T L I R I W G D A L F D I V D K D Q N G A I T L DE W K A Y T K A A G I I Q S S E D C E E T F R V C D I D E S G Q L D V DE M T R Q H L G F W Y T M D P A C E K L Y G G A V P G2A M S K G E E L FT G V V P I L V E L D G D V N G H K F S V S G E G E G D A T [SEQ ID NO:3] Y G K L T L K F I C T T G K L P V P W P T L V T T L T Y G V Q C F S RY P D H M K Q H D F F K S A M P E G Y V Q E R T I F F K D D G N Y K T RA E V K F E G D T L V N R I E L K G I D F K E D G N I L G H K L E Y N YN S H N V Y I M A D K Q K N G I K A N F K I R H N I E D G S V Q L A D HY Q Q N T P I G D G P V L L P D N H Y L S T Q S A L S K D P N E K R D HM V L L E F V T A A G I T H G M D E L Y K S G G S G S G G Q S G G S G SG G Q S G L R S V K L T S D F D N P R W I G R H K H M F N F L D V N H NG K I S L D E M V Y K A S D I V I N N L G A T P E Q A K R H K D A V E AF F G G A G M K Y G V E T D W P A Y I E G W K K L A T D E L E K Y A K NE P T L I R I W G D A L F D I V D K D Q N G A I T L D E W K A Y T K A AG I I Q S S E D C E E T F R V C D I D E S G Q L D V D E M T R Q H L G FW Y T M D P A C E K L Y G G A V P G4A M S K G E E L F T G V V P I L V EL D G D V N G H K F S V S G E G E G D A T [SEQ ID NO: 4] Y G K L T L K FI C T T G K L P V P W P T L V T T L T Y G V Q C F S R Y P D H M K Q H DF F K S A M P E G Y V Q E R T I F F K D D G N Y K T R A E V K F E G D TL V N R I E L K G I D F K E D G N I L G H K L E Y N Y N S H N V Y I M AD K Q K N G I K A N F K I R H N I E D G S V Q L A D H Y Q Q N T P I G DG P V L L P D N H Y L S T Q S A L S K D P N E K R D H M V L L E F V T AA G I T H G M D E L Y K S G G S G S G G Q S G G S G S G G Q S G G S G SG G Q S G G S G S G G Q S G L R S V K L T S D F D N P R W I G R H K H MF N F L D V N H N G K I S L D E M V Y K A S D I V I N N L G A T P E Q AK R H K D A V E A F F G G A G M K Y G V E T D W P A Y I E G W K K L A TD E L E K Y A K N E P T L I R I W G D A L F D I V D K D Q N G A I T L DE W K A Y T K A A G I I Q S S E D C E E T F R V C D I D E S G Q L D V DE M T R Q H L G F W Y T M D P A C E K L Y G G A V P G5A M S K G E E L FT G V V P I L V E L D G D V N G H K F S V S G E G E G D A T [SEQ ID NO:5] Y G K L T L K F I C T T G K L P V P W P T L V T T L T Y G V Q C F S RY P D H M K Q H D F F K S A M P E G Y V Q E R T I F F K D D G N Y K T RA E V K F E G D T L V N R I E L K G I D F K E D G N I L G H K L E Y N YN S H N V Y I M A D K Q K N G I K A N F K I R H N I E D G S V Q L A D HY Q Q N T P I G D G P V L L P D N H Y L S T Q S A L S K D P N E K R D HM V L L E F V T A A G I T H G M D E L Y K S G G S G S G G Q S G G S G SG G Q S G G S G S G G Q S G G S G S G G Q S G G S G S G G Q S G L R S VK L T S D F D N P R W I G R H K H M F N F L D V N H N G K I S L D E M VY K A S D I V I N N L G A T P E Q A K R H K D A V E A F F G G A G M K YG V E T D W P A Y I E G W K K L A T D E L E K Y A K N E P T L I R I W GD A L F D I V D K D Q N G A I T L D E W K A Y T K A A G I I Q S S E D CE E T F R V C D I D E S G Q L D V D E M T R Q H L G F W Y T M D P A C EK L Y G G A V P SeG5A M V S A S R P E A L A A P V T T V A T L V P H N AT E P A S P G E G K E D A [SEQ ID NO: 6) F S K L K Q K F M N E L H K I PL P P W A L I A I A I V A V L L V V T C C F C V C K K C L F K K K N K KK G K E K G G K N A I N M K D V K D L G K T M K D Q A L K D D D A E T GL T D G E E K E E P K E E E K L G K L Q Y S L D Y D F Q N N Q L L V G II Q A A E L P A L D M G G T S D P Y V K V F L L P D K K K K F E T K V HR K T L N P V F N E Q F T F K V P Y S E L G G K T L V M A V Y D F D R FS K H D I I G E F K V P M N T V D F G H V T E E W R D L Q S A E K E E QE K L G D I C F S L R Y V P T A G K L T V V I L E A K N L K K M D V G GL S D P Y V K I H L M Q N G K R L K K K K T T I K K N T L N P Y Y N E SF S F E V P F E Q I Q K V Q V V V T V L D Y D K I G K N D A I G K V F VG Y N S T G A E L R H W S D M L A N P R R P I A Q W H T L Q V E E E V DA M L A V K R S G N S G R A T M S K G E E L F T G V V P I L V E L D G DV N G H K F S V S G E G E G D A T Y G K L T L K F I C T T G K L p V P WP T L V T T L T Y G V Q C F S R Y P D H M K Q H D F F K S A M P E G Y VQ E R T I F F K D D G N Y K T R A E V K F E G D T L V N R I E L K G I DF K E D G N I L G H K L E Y N Y N S H N V Y I M A D K Q K N G I K A N FK I R H N I E D G S V Q L A D H Y Q Q N T P I G D G P V L L P D N H Y LS T Q S A L S K D P N E K R D H M V L L E F V T A A G I T H G M D E L YK S G G S G S G G Q S G G S G S G G Q S G G S G S G G Q S G G S G S G GQ S G G S G S G G Q S G L R S V K L T S D F D N P R W I G R H K H M F NF L D V N H N G K I S L D E M V Y K A S D I V I N N L G A T P E Q A K RH K D A V E A F F G G A G M K Y G V E T D W P A Y I E G W K K L A T D EL E K Y A K N E P T L I R I W G D A L F D I V D K D Q N G A I T L D E WK A Y T K A A G I I Q S S E D C E E T F R V C D I D E S G Q L D V D E MT R Q H L G F W Y T M D P A C E K L Y G G A V P GA Atg agc aag ggc gaggag ctg ttc acc ggg gtg gtg ccc atc ctg gtc gag ctg gac [SEQ ID NO: 7]ggc gac gta aac ggc cac aag ttc agc gtg tcc ggc gag ggc gag ggc gat gccacc tac ggc aag ctg acc ctg aag ttc atc tgc acc acc ggc aag ctg ccc gtgccc tgg ccc acc ctc gtg acc acc ctg acc tac ggc gtg cag tgc ttc agc cgctac ccc gac cac atg aag cag cac gac ttc ttc aag tcc gcc atg ccc gaa ggctac gtc cag gag cgc acc atc ttc ttc aag gac gac ggc aac tac aag acc cgcgcc gag gtg aag ttc gag ggc gac acc ctg gtg aac cgc atc gag ctg aag ggcatc gac ttc aag gag gac ggc aac atc ctg ggg cac aag ctg gag tac aac tacaac agc cac aac gtc tat atc atg gcc gac aag cag aag aac ggc atc aag gCCaac ttc aag atc cqc cac aac atc gag gac ggc agc gtg cag ctc qcc gac cactac cag cag aac acc ccc atc ggc gac ggc ccc gtg ctg ctg ccc gac aac cactac ctg agc acc cag tcc gcc ctg agc aaa gac ccc aac gag aag cgc gat cacatg gtc ctg ctg gag ttc gtg acc gcc gcc ggg atc act cAc ggc atg gac gagctg tac aag tcc gga ctc aGA TCT gtc aaa ctt aca tca gac ttc gac aac ccaaga tgg att gga cga cac aag cat atg ttc aat ttc ctt gat gtc aac cac aatgga aaa atc tct ctt gac gag atg gtc tac aag gca tct gat att gtc atc aataac ctt gga gca aca cct gag caa gcc aaa cga cac aaa gat gct gtG gaa gccttc ttc gga gga gct gga atg aaa tat ggt gtg gaa act gat tgg cct gca tatatt gaa gga tgg aaa aaa ttg gct act gat gaa ttg gag aaa tac gcc aaa aacgaa cca acC ctc atc cgC ata tgg ggt gat gct ttg ttt gat atc gtt gac aaagat caa aat gga gct att aca ctg gat gaa tgg aaa gca tac acc aaa gct gctggt atc atc caa tca tca gaa gat tgc gag gaa aca ttc aga gtg tgc gat attgat gaa agt gga caa ctc gat gtt gat gag atg aca aga caG cat CtG gga ttttgg tac acc atg gat cct gct tgc gaa aag ctc tac ggt gga gct gtc ccc G1AAtg agc aag ggo gag gag ctg ttc acc ggg gtg gtg ccc atc ctg gtc gag ctggac [SEQ ID NO: 8] ggc gac gta aac ggc cac aag ttc agc gtg tcc ggc gagggc gag ggc gat gcc acc tac ggc aag ctg acc ctg aag ttc atc tgc acc accggc aag ctg ccc gtg ccc tgg ccc acc ctc gtg acc acc ctg acc tac ggc gtgcag tgc ttc agc cgc tac ccc gac cac atg aag cag cac gac ttc ttc aag tccgcc atg ccc gaa ggc tac gtc cag gag cgc acc atc ttc ttc aag gac gac ggcaac tac aag acc cgc gcc gag gtg aag ttc gag ggc gac acc ctg gtg aac cgcatc gag ctg aag ggc atc gac ttc aag gag gac ggc aac atc ctg ggg cac aagctg gag tac aac tac aac agc cac aac gtc tat atc atg gcc gac aag cag aagaac ggc atc aag gCC aac ttc aag atc cgc cac aac atc gag gac ggc agc gtgcag ctc gcc gac cac tac cag cag aac acc ccc atc ggc gac ggc ccc gtg ctgctg ccc gac aac cac tac ctg agc acc cag tcc gcc ctg agc aaa gac ccc aacgag aag cgc gat cac atg gtc ctg ctg gag ttc gtg acc gcc gcc ggg atc actcAc ggc atg gac gag ctg tac aag tcc ggc ggg agc gga tcc ggc ggc cag tccggc ctc aGA TCT gtc aaa ctt aca tca gac ttc gac aac cca aga tgg att ggacga cac aag cat atg ttc aat ttc ctt gat gtc aac cac aat gga aaa atc tctctt gac gag atg gtc tac aag gca tct gat att gtc atc aat aac ctt gga gcaaca cct gag caa gcc aaa cga cac aaa gat gct gtG gaa gcc ttc ttc gga ggagct gga atg aaa tat ggt gtg gaa act gat tgg cct gca tat att gaa gga tggaaa aaa ttg gct act gat gaa ttg gag aaa tac gcc aaa aac gaa cca acC ctcatc cgC ata tgg ggt gat gct ttg ttt gat atc gtt gac aaa gat caa aat ggagct att aca ctg gat gaa tgg aaa gca tac acc aaa gct gct ggt atc atc caatca tca gaa gat tgc gag gaa aca ttc aga gtg tgc gat att gat gaa agt ggacaa ctc gat gtt gat gag atg aca aga caG cat CtG gga ttt tgg tac acc atggat cct gct tgc gaa aag ctc tac ggt gga gct gtc ccc G2A Atg agc aag ggcgag gag ctg ttc acc ggg gtg gtg ccc atc ctg gtc gag ctg gac [SEQ ID NO:9] ggc gac gta aac ggc cac aag ttc agc gtg tcc ggc gag ggc gag ggc gatgcc acc tac ggc aag ctg acc ctg aag ttc atc tgc acc acc ggc aag ctg cccgtg ccc tgg ccc acc ctc gtg acc acc ctg acc tac ggc gtg cag tgc ttc agccgc tac ccc gac cac atg aag cag cac gac ttc ttc aag tcc gcc atg ccc gaaggc tac gtc cag gag cgc acc atc ttc tcc aag gac gac ggc aac tac aag acccgc gcc gag gtg aag ttc gag ggc gac acc ctg gtg aac cgc atc gag ctg aagggc atc gac ttc aag gag gac ggc aac atc ctg ggg cac aag ctg gag tac aactac aac agc cac aac gtc tac atc atg gcc gac aag cag aag aac ggc atc aaggCC aac ttc aag atc cgc cac aac atc gag gac ggc agc gtg cag ctc gcc gaccac tac cag cag aac acc ccc atc ggc gac ggc ccc gtg ctg ctg ccc gac aaccac tac ctg agc acc cag tcc gcc ctg agc aaa gac ccc aac gag aag cgc gatcac atg gtc ctg ctg gag ttc gtg acc gcc gcc ggg atc act cAc ggc atg gacgag ctg tac aag tcc ggc ggg agc gga tcc ggc ggc cag tcc ggc ggg agc ggatcc ggc ggc cag tcc ggc ctc aGA TCT gtc aaa ctt aca tca gac ttc gac aaccca aga tgg att gga cga cac aag cat atg ttc aat ttc ctt gat gtc aac cacaat gga aaa atc tct ctt gac gag atg gtc tac aag gca tct gat att gtc atcaat aac ctt gga gca aca cct gag caa gcc aaa cga cac aaa gat gct gtG gaagcc ttc ttc gga gga gct gga atg aaa tat ggt gtg gaa act gat tgg cct gcatat att gaa gga tgg aaa aaa ttg gct act gat gaa ttg gag aaa tac gcc aaaaac gaa cca acC ctc atc cgC ata tgg ggt gat gct ttg ttt gat atc gtt gacaaa gat caa aat gga gct att aca ctg gat gaa tgg aaa gca tac acc aaa gctgct ggt atc atc caa tca tca gaa gat tgc gag gaa aca ttc aga gtg tgc gatatt gat gaa agt gga caa ctc gat gtt gat gag atg aca aga caG cat CtG ggattt tgg tac acc atg gat cct gct tgc gaa aag ctc tac ggt gga gct gtc cccG4A Atg agc aag ggc gag gag ctg ttc acc ggg gtg gtg ccc atc ctg gtc gagctg gac [SEQ ID NO: 10] ggc gac gta aac ggc cac aag ttc agc gtg tcc ggcgag ggc gag ggc gat gcc acc tac ggc aag ctg acc ctg aag ttc atc tgc accacc ggc aag ctg ccc gtg ccc tgg ccc acc ctc gtg acc acc ctg acc tac ggcgtg cag tgc ttc agc cgc tac ccc gac cac atg aag cag cac gac ttc ttc aagtcc gcc atg ccc gaa ggc tac gtc cag gag cgc acc atc ttc ttc aag gac gacggc aac tac aag acc cgc gcc gag gtg aag ttc gag ggc gac acc ctg gtg aaccgc atc gag ctg aag ggc atc gac ttc aag gag gac ggc aac atc ctg ggg cacaag ctg gag tac aac tac aac agc cac aac gtc tat atc atg gcc gac aag cagaag aac ggc atc aag gCC aac ttc aag atc cgc cac aac atc gag gac ggc agcgtg cag ctc gcc gac cac tac cag cag aac acc ccc atc ggc gac ggc ccc gtgctg ctg ccc gac aac cac tac ctg agc acc cag tcc gcc ctg agc aaa gac cccaac gag aag cgc gat cac atg gtc ctg ctg gag ttc gtg acc gcc gcc ggg atcact cAc ggc atg gac gag ctg tac aag tcc ggc ggg agc gga tcc ggc ggc cagtcc ggc ggg agc gga tcc ggc ggc cag tcc ggc ggg agc gga tcc ggc ggc cagtcc ggc ggg agc gga tcc ggc ggc cag tcc ggc ctc aGA TCT gtc aaa ctt acatca gac ttc gac aac cca aga tgg att gga cga cac aag cat atg ttc aat ttcctt gat gtc aac cac aat gga aaa atc tct ctt gac gag atg gtc tac aag gcatct gat att gtc atc aat aac ctt gga gca aca cct gag caa gcc aaa cga cacaaa gat gct gtG gaa gcc ttc ttc gga gga gct gga atg aaa tat ggt gtg gaaact gat tgg cct gca tat att gaa gga tgg aaa aaa ttg gct act gat gaa ttggag aaa tac gcc aaa aac gaa cca acC ctc atc cgC ata tgg ggt gat gct ttgttt gat atc gtt gac aaa gat caa aat qga gct att aca ctg gat gaa tgg aaagca tac acc aaa gct gct ggt atc atc caa tca tca gaa gat tgc gag gaa acattc aga gtg tgc gat att gat gaa agt gga caa ctc gat gtt gat gag atg acaaga caG cat CtG gga ttt tgg tac acc atg gat ccc gct tgc gaa aag ctc tacggt gga gct gtc ccc G5A Atg agc aag ggc gag gag ctg ttc acc ggg gtg gtgccc atc ctg gtc gag ctg gac [SEQ ID NO: 11] ggc gac gta aac ggc cac aagttc agc gtg tcc ggc gag ggc gag ggc gat gcc acc tac ggc aag ctg acc ctgaag ttc atc tgc acc acc ggc aag ctg ccc gtg ccc tgg ccc acc ctc gtg accacc ctg acc tac ggc gtg cag tgc ttc agc cgc tac ccc gac cac atg aag cagcac gac ttc ttc aag tcc gcc atg ccc gaa ggc tac gtc cag gag cgc acc atcttc ttc aag gac gac ggc aac tac aag acc cgc gcc gag gtg aag ttc gag ggcgac acc ctg gtg aac cgc atc gag ctg aag ggc atc gac ttc aag gag gac ggcaac atc ctg ggg cac aag ctg gag tac aac tac aac agc cac aac gtc tat atcatg gcc gac aag cag aag aac ggc atc aag gCC aac ttc aag atc cgc cac aacatc gag gac ggc agc gtg cag ctc gcc gac cac tac cag cag aac acc ccc atcggc gac ggc ccc gtg ctg ctg ccc gac aac cac tac ctg agc acc cag tcc gccctg agc aaa gac ccc aac gag aag cgc gat cac atg gtc ctg ctg gag ttc gtgacc gcc gcc ggg atc act cAc ggc atg gac gag ctg tac aag tcc ggc ggg agcgga tcc ggc ggc cag tcc ggc ggg agc gga tcc ggc ggc cag tcc ggc ggg agcgga tcc ggc ggc cag tcc ggc ggg agc gga tcc ggc ggc cag tcc ggc ggg agcgga tcc ggc ggc cag tcc ggc ctc aGA TCT gtc aaa ctt aca tca gac ttc gacaac cca aga tgg att gga cga cac aag cat atg ttc aat ttc ctt gat gtc aaccac aat gga aaa atc tct ctt gac gag atg gtc tac aag gca tct gat att gtcatc aat aac ctt gga gca aca cct gag caa gcc aaa cga cac aaa gat gct gtGgaa gcc ttc ttc gga gga gct gga atg aaa tat ggt gtg gaa act gat tgg cctgca tat att gaa gga tgg aaa aaa ttg gct act gat gaa ttg gag aaa tac gccaaa aac gaa cca acC ctc atc cgC ata tgg ggt gat gct ttg ttt gat atc gttgac aaa gat caa aat gga gct att aca ctg gat gaa tgg aaa gca tac acc aaagct gct ggt atc atc caa tca tca gaa gat tgc gag gaa aca ttc aga gtg tgcgat att gat gaa aqt gga caa ctc gat gtt gat gag atg aca aga caG cat CtGgga ttt tgg tac acc atg gat cct gct tgc gaa aag ctc tac ggt gga gct gtcccc SeG5A Atg gtg agt gcc agt cgt cct gag gcc ctg gct gcc cct gtc accact gtt gcg acc [SEQ ID NO: 12] ctt gtc cca cac aac gcc act gag cca gccagt cct ggg gaa ggg aag gaa gat gcc ttt tcc aag ctg aag cag aag ttt atgaat gaa ctg cat aaa atc cca ttg cca ccg tgg gcc tta att gcc ata gcc atagtt gcg gtc ctt cta gtc gtg acc tgc tgc ttc tgt gtc tgt aag aaa tgt ttgttc aaa aag aaa aac aag aag aag gga aag gaa aag gga ggg aag aac gcc attaac atg aaa gac gtg aaa gac tta ggg aag acc atg aag gat cag gcc ctt aaggat gac gat gct gaa act gga ctg act gat gga gaa gaa aag gag gag ccc aaggaa gag gag aaa ctg gga aag ctt caa tat tca ctg gac tat gac ttc cag aataac cag ctg ctg gtg gga atc atc cag gct gct gaa ctg ccc gcc ctg gac atggga ggc aca tct gat cca tac gtc aaa gtc ttc ctg ctg ccc gac aaa aag aagaag ttt gag aca aaa gtc cac cgg aaa acc ctc aat cca gtc ttc aat gaa cagttt act ttc aag gtg cca tac tcg gaa tta ggt ggc aag aca ctg gtg atg gctgtg tat gat ttt gac cgc ttc tcc aag cac gac atc att gga gag ttc aaa gttcct atg aac acc gtg gat ttt ggc cac gtc acc gag gag tgg cgc gat ctc cagagt gct gag aaa gaa gag caa gag aaa ctg ggt gac atc tgc ttc tcc ctc cgctac gtc cct act gcc ggc aag ctg act gtt gtc att ctg gaa gcc aag aac ctgaag aag atg gat gtg qgt ggc tta tct gat ccc tat gta aag att cac ctg atgcag aac ggc aag aga ctg aag aag aaa aag aca acg att aag aag aac aca cttaac ccc tac tac aat gag tcc ttc agc ttt gaa gtt ccg ttc gag caa atc cagaaa gtg caa gtg gtg gta act gtt ttg gac tat gac aag att ggc aag aac gacgcc atc ggc aaa gtc ttt gtg ggc tac aac agc acc ggc gca gag ctg cga cactgg tca gac atg ctg gcc aac ccc cgg cgc ccc atc gcc cag tgg cac act ctgcag gta gag gag gag gtt gat gcc atg ctg gct gtc aag aGA tCC GGG AAT TCCGGG CGG gcc acc atg agc aag ggc gag gag ctg ttc acc ggg gtg gtg ccc atcctg gtc gag ctg gac ggc gac gta aac ggc cac aag ttc agc gtg tcc ggc gagggc gag ggc gat gcc acc tac ggc aag ctg acc ctg aag ttc atc tgc acc accggc aag ctg ccc gtg ccc tgg ccc acc ctc gtg acc acc ctg acc tac ggc gtgcag tgc ttc agc cgc tac ccc gac cac atg aag cag cac gac ttc ttc aag tccgcc atg ccc gaa ggc tac gtc cag gag cgc acc atc ttc ttc aag gac gac ggcaac tac aag acc cgc gcc gag gtg aag ttc gag ggc gac acc ctg gtg aac cgcatc gag ctg aag ggc atc gac ttc aag gag gac ggc aac atc ctg ggg cac aagctg gag tac aac tac aac agc cac aac gtc tat atc atg gcc gac aag cag aagaac ggc atc aag gCC aac ttc aag atc cgc cac aac atc gag gac ggc agc gtgcag ctc gcc gac cac tac cag cag aac acc ccc atc ggc gac ggc ccc gtg ctqctg ccc gac aac cac tac ctg agc acc cag tcc gcc ctg agc aaa gac ccc aacgag aag cgc gat cac atg gtc ctg ctg gag ttc gtg acc gcc gcc ggg atc actcAc ggc atg gac gag ctg tac aag tcc ggc ggg agc gga tcc ggc ggc cag tccggc ggg agc gga tcc ggc ggc cag tcc ggc ggg agc gga tcc ggc ggc cag tccggc ggg agc gga tcc ggc ggc cag tcc ggc ggg agc gga tcc ggc ggc cag tccggc ctc aGA TCT gtc aaa ctt aca tca gac ttc gac aac cca aga tgg att ggacga cac aag cat atg ttc aat ttc ctt gat gtc aac cac aat gga aaa atc tctctt gac gag atg gtc tac aag gca tct gat att gtc atc aat aac ctt gga gcaaca cct gag caa gcc aaa cga cac aaa gat gct gtG gaa gcc ttc ttc gga ggagct gga atg aaa tat ggt gtg gaa act gat tgg cct gca tat att gaa gga tggaaa aaa ttg gct act gat gaa ttg gag aaa tac gcc aaa aac gaa cca acC ctcatc cgC ata tgg ggt gat gct ttg ttt gat atc gtt gac aaa gat caa aat ggagct att aca ctg gat gaa tgg aaa gca tac acc aaa gct gct ggt atc atc caatca tca gaa gat tgc gag gaa aca ttc aga gtq tgc gat att gat gaa agt ggacaa ctc gat gtt gat gag atg aca aga caG cat CtG gga ttt tgg tac acc atggat cct gct tgc gaa aag ctc tac ggt gga gct gtc ccc DNA sequence ofGFP-aeguorin linkers pGA (strain I2507)      TCC GGC CTC AGA TCT [SEQ IDNO: 13] pG1A (strain I2508)     TCC GGC GGG AGC GGA TCC GGC GGC CAG TCC[SEQ ID NO: 14]                         GGC CTC AGA TCT pG2A (strainI2509)     TCC GGC GGG AGC GGA TCC GGC GGC CAG TCC [SEQ ID NO: 15]                        GGC GGG AGC GGA TCC GGC GGC CAG TCC GGC CTC                        AGA TCT pG4A (strain I2510)     TCC GGC GGG AGCGGA TCC GGC GGC CAG TCC [SEQ ID NO: 16]                         GGC GGGAGC GGA TCC GGC GGC CAG TCC GGC GGG                         AGC GGA TCCGGC GGC CAG TCC GGC GGG AGC GGA                         TCC GGC GGC CAGTCC GGC CTC AGA TCT pG5A (strain I2511)     TCC GGC GGG AGC GGA TCC GGCGGC CAG TCC [SEQ ID NO: 17]                         GGC GGG AGC GGA TCCGGC GGC CAG TCC GGC GGG                         AGC GGA TCC GGC GGC CAGTCC GGC GGG AGC GGA                         TCC GGC GGC CAG TCC GGC GGGAGC GGA TCC GGC                         GGC CAG TCC GGC CTC AGA TCTpSeG5A (strain I2512) and pStG5A (strain I2513)same linker sequence thanpG5A. Peptide sequence of linkers pGA Ser Gly Leu Arg Ser [SEQ ID NO:18] Pg1a Ser Gly Gly Ser Gly Ser Gly Gly Gln Ser Gly Leu Arg Ser [SEQ IDNO: 19] pG2A Ser Gly Gly Ser Gly Ser Gly Gly Gln Ser Gly Gly Ser Gly[SEQ ID NO: 20] Ser Gly Gly Gln Ser Gly Leu Arg Ser pG4A Ser Gly Gly SerGly Ser Gly Gly Gln Ser Gly Gly Ser Gly [SEQ ID NO: 21] Ser Gly Gly GlnSer Gly Gly Ser Gly Ser Gly Gly Gln Ser Gly Gly Ser Gly Ser Gly Gly GlnSer Gly Leu Arg Ser pG5A Ser Gly Gly Ser Gly Ser Gly Gly Gln Ser Gly GlySer Gly [SEQ ID NO: 22] Ser Gly Gly Gln Ser Gly Gly Ser Gly Ser Gly GlyGln Ser Gly Gly Ser Gly Ser Gly Gly Gln Ser Gly Gly Ser Gly Ser Gly GlyGln Ser Gly Leu Arg Ser pSeG5A and pStGSA idem than pG5A.

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Also incorporated by reference herein in its entirety is U.S. Pat. No.5,683,888.

48 1 432 PRT Aequorea victoria 1 Met Ser Lys Gly Glu Glu Leu Phe Thr GlyVal Val Pro Ile Leu Val 1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly HisLys Phe Ser Val Ser Gly Glu 20 25 30 Gly Glu Gly Asp Ala Thr Tyr Gly LysLeu Thr Leu Lys Phe Ile Cys 35 40 45 Thr Thr Gly Lys Leu Pro Val Pro TrpPro Thr Leu Val Thr Thr Leu 50 55 60 Thr Tyr Gly Val Gln Cys Phe Ser ArgTyr Pro Asp His Met Lys Gln 65 70 75 80 His Asp Phe Phe Lys Ser Ala MetPro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp Asp GlyAsn Tyr Lys Thr Arg Ala Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr LeuVal Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 Asp Phe Lys Glu Asp GlyAsn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 Tyr Asn Ser His AsnVal Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 Ile Lys AlaAsn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175 Gln LeuAla Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 ValLeu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215220 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys Ser Gly 225230 235 240 Leu Arg Ser Val Lys Leu Thr Ser Asp Phe Asp Asn Pro Arg TrpIle 245 250 255 Gly Arg His Lys His Met Phe Asn Phe Leu Asp Val Asn HisAsn Gly 260 265 270 Lys Ile Ser Leu Asp Glu Met Val Tyr Lys Ala Ser AspIle Val Ile 275 280 285 Asn Asn Leu Gly Ala Thr Pro Glu Gln Ala Lys ArgHis Lys Asp Ala 290 295 300 Val Glu Ala Phe Phe Gly Gly Ala Gly Met LysTyr Gly Val Glu Thr 305 310 315 320 Asp Trp Pro Ala Tyr Ile Glu Gly TrpLys Lys Leu Ala Thr Asp Glu 325 330 335 Leu Glu Lys Tyr Ala Lys Asn GluPro Thr Leu Ile Arg Ile Trp Gly 340 345 350 Asp Ala Leu Phe Asp Ile ValAsp Lys Asp Gln Asn Gly Ala Ile Thr 355 360 365 Leu Asp Glu Trp Lys AlaTyr Thr Lys Ala Ala Gly Ile Ile Gln Ser 370 375 380 Ser Glu Asp Cys GluGlu Thr Phe Arg Val Cys Asp Ile Asp Glu Ser 385 390 395 400 Gly Gln LeuAsp Val Asp Glu Met Thr Arg Gln His Leu Gly Phe Trp 405 410 415 Tyr ThrMet Asp Pro Ala Cys Glu Lys Leu Tyr Gly Gly Ala Val Pro 420 425 430 2441 PRT Aequorea victoria 2 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly ValVal Pro Ile Leu Val 1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly His LysPhe Ser Val Ser Gly Glu 20 25 30 Gly Glu Gly Asp Ala Thr Tyr Gly Lys LeuThr Leu Lys Phe Ile Cys 35 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp ProThr Leu Val Thr Thr Leu 50 55 60 Thr Tyr Gly Val Gln Cys Phe Ser Arg TyrPro Asp His Met Lys Gln 65 70 75 80 His Asp Phe Phe Lys Ser Ala Met ProGlu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp Asp Gly AsnTyr Lys Thr Arg Ala Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr Leu ValAsn Arg Ile Glu Leu Lys Gly Ile 115 120 125 Asp Phe Lys Glu Asp Gly AsnIle Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 Tyr Asn Ser His Asn ValTyr Ile Met Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 Ile Lys Ala AsnPhe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175 Gln Leu AlaAsp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 Val LeuLeu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205 LysAsp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215 220Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys Ser Gly 225 230235 240 Gly Ser Gly Ser Gly Gly Gln Ser Gly Leu Arg Ser Val Lys Leu Thr245 250 255 Ser Asp Phe Asp Asn Pro Arg Trp Ile Gly Arg His Lys His MetPhe 260 265 270 Asn Phe Leu Asp Val Asn His Asn Gly Lys Ile Ser Leu AspGlu Met 275 280 285 Val Tyr Lys Ala Ser Asp Ile Val Ile Asn Asn Leu GlyAla Thr Pro 290 295 300 Glu Gln Ala Lys Arg His Lys Asp Ala Val Glu AlaPhe Phe Gly Gly 305 310 315 320 Ala Gly Met Lys Tyr Gly Val Glu Thr AspTrp Pro Ala Tyr Ile Glu 325 330 335 Gly Trp Lys Lys Leu Ala Thr Asp GluLeu Glu Lys Tyr Ala Lys Asn 340 345 350 Glu Pro Thr Leu Ile Arg Ile TrpGly Asp Ala Leu Phe Asp Ile Val 355 360 365 Asp Lys Asp Gln Asn Gly AlaIle Thr Leu Asp Glu Trp Lys Ala Tyr 370 375 380 Thr Lys Ala Ala Gly IleIle Gln Ser Ser Glu Asp Cys Glu Glu Thr 385 390 395 400 Phe Arg Val CysAsp Ile Asp Glu Ser Gly Gln Leu Asp Val Asp Glu 405 410 415 Met Thr ArgGln His Leu Gly Phe Trp Tyr Thr Met Asp Pro Ala Cys 420 425 430 Glu LysLeu Tyr Gly Gly Ala Val Pro 435 440 3 450 PRT Aequorea victoria 3 MetSer Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu 50 55 60Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln 65 70 7580 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 9095 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu TyrAsn 130 135 140 Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln LysAsn Gly 145 150 155 160 Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile GluAsp Gly Ser Val 165 170 175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr ProIle Gly Asp Gly Pro 180 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu SerThr Gln Ser Ala Leu Ser 195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp HisMet Val Leu Leu Glu Phe Val 210 215 220 Thr Ala Ala Gly Ile Thr His GlyMet Asp Glu Leu Tyr Lys Ser Gly 225 230 235 240 Gly Ser Gly Ser Gly GlyGln Ser Gly Gly Ser Gly Ser Gly Gly Gln 245 250 255 Ser Gly Leu Arg SerVal Lys Leu Thr Ser Asp Phe Asp Asn Pro Arg 260 265 270 Trp Ile Gly ArgHis Lys His Met Phe Asn Phe Leu Asp Val Asn His 275 280 285 Asn Gly LysIle Ser Leu Asp Glu Met Val Tyr Lys Ala Ser Asp Ile 290 295 300 Val IleAsn Asn Leu Gly Ala Thr Pro Glu Gln Ala Lys Arg His Lys 305 310 315 320Asp Ala Val Glu Ala Phe Phe Gly Gly Ala Gly Met Lys Tyr Gly Val 325 330335 Glu Thr Asp Trp Pro Ala Tyr Ile Glu Gly Trp Lys Lys Leu Ala Thr 340345 350 Asp Glu Leu Glu Lys Tyr Ala Lys Asn Glu Pro Thr Leu Ile Arg Ile355 360 365 Trp Gly Asp Ala Leu Phe Asp Ile Val Asp Lys Asp Gln Asn GlyAla 370 375 380 Ile Thr Leu Asp Glu Trp Lys Ala Tyr Thr Lys Ala Ala GlyIle Ile 385 390 395 400 Gln Ser Ser Glu Asp Cys Glu Glu Thr Phe Arg ValCys Asp Ile Asp 405 410 415 Glu Ser Gly Gln Leu Asp Val Asp Glu Met ThrArg Gln His Leu Gly 420 425 430 Phe Trp Tyr Thr Met Asp Pro Ala Cys GluLys Leu Tyr Gly Gly Ala 435 440 445 Val Pro 450 4 468 PRT Aequoreavictoria 4 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile LeuVal 1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val SerGly Glu 20 25 30 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys PheIle Cys 35 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val ThrThr Leu 50 55 60 Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His MetLys Gln 65 70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr ValGln Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr ArgAla Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile GluLeu Lys Gly Ile 115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly HisLys Leu Glu Tyr Asn 130 135 140 Tyr Asn Ser His Asn Val Tyr Ile Met AlaAsp Lys Gln Lys Asn Gly 145 150 155 160 Ile Lys Ala Asn Phe Lys Ile ArgHis Asn Ile Glu Asp Gly Ser Val 165 170 175 Gln Leu Ala Asp His Tyr GlnGln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 Val Leu Leu Pro Asp AsnHis Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205 Lys Asp Pro Asn GluLys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215 220 Thr Ala Ala GlyIle Thr His Gly Met Asp Glu Leu Tyr Lys Ser Gly 225 230 235 240 Gly SerGly Ser Gly Gly Gln Ser Gly Gly Ser Gly Ser Gly Gly Gln 245 250 255 SerGly Gly Ser Gly Ser Gly Gly Gln Ser Gly Gly Ser Gly Ser Gly 260 265 270Gly Gln Ser Gly Leu Arg Ser Val Lys Leu Thr Ser Asp Phe Asp Asn 275 280285 Pro Arg Trp Ile Gly Arg His Lys His Met Phe Asn Phe Leu Asp Val 290295 300 Asn His Asn Gly Lys Ile Ser Leu Asp Glu Met Val Tyr Lys Ala Ser305 310 315 320 Asp Ile Val Ile Asn Asn Leu Gly Ala Thr Pro Glu Gln AlaLys Arg 325 330 335 His Lys Asp Ala Val Glu Ala Phe Phe Gly Gly Ala GlyMet Lys Tyr 340 345 350 Gly Val Glu Thr Asp Trp Pro Ala Tyr Ile Glu GlyTrp Lys Lys Leu 355 360 365 Ala Thr Asp Glu Leu Glu Lys Tyr Ala Lys AsnGlu Pro Thr Leu Ile 370 375 380 Arg Ile Trp Gly Asp Ala Leu Phe Asp IleVal Asp Lys Asp Gln Asn 385 390 395 400 Gly Ala Ile Thr Leu Asp Glu TrpLys Ala Tyr Thr Lys Ala Ala Gly 405 410 415 Ile Ile Gln Ser Ser Glu AspCys Glu Glu Thr Phe Arg Val Cys Asp 420 425 430 Ile Asp Glu Ser Gly GlnLeu Asp Val Asp Glu Met Thr Arg Gln His 435 440 445 Leu Gly Phe Trp TyrThr Met Asp Pro Ala Cys Glu Lys Leu Tyr Gly 450 455 460 Gly Ala Val Pro465 5 477 PRT Aequorea victoria 5 Met Ser Lys Gly Glu Glu Leu Phe ThrGly Val Val Pro Ile Leu Val 1 5 10 15 Glu Leu Asp Gly Asp Val Asn GlyHis Lys Phe Ser Val Ser Gly Glu 20 25 30 Gly Glu Gly Asp Ala Thr Tyr GlyLys Leu Thr Leu Lys Phe Ile Cys 35 40 45 Thr Thr Gly Lys Leu Pro Val ProTrp Pro Thr Leu Val Thr Thr Leu 50 55 60 Thr Tyr Gly Val Gln Cys Phe SerArg Tyr Pro Asp His Met Lys Gln 65 70 75 80 His Asp Phe Phe Lys Ser AlaMet Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp AspGly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 Lys Phe Glu Gly Asp ThrLeu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 Asp Phe Lys Glu AspGly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 Tyr Asn Ser HisAsn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 Ile LysAla Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175 GlnLeu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val 210215 220 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys Ser Gly225 230 235 240 Gly Ser Gly Ser Gly Gly Gln Ser Gly Gly Ser Gly Ser GlyGly Gln 245 250 255 Ser Gly Gly Ser Gly Ser Gly Gly Gln Ser Gly Gly SerGly Ser Gly 260 265 270 Gly Gln Ser Gly Gly Ser Gly Ser Gly Gly Gln SerGly Leu Arg Ser 275 280 285 Val Lys Leu Thr Ser Asp Phe Asp Asn Pro ArgTrp Ile Gly Arg His 290 295 300 Lys His Met Phe Asn Phe Leu Asp Val AsnHis Asn Gly Lys Ile Ser 305 310 315 320 Leu Asp Glu Met Val Tyr Lys AlaSer Asp Ile Val Ile Asn Asn Leu 325 330 335 Gly Ala Thr Pro Glu Gln AlaLys Arg His Lys Asp Ala Val Glu Ala 340 345 350 Phe Phe Gly Gly Ala GlyMet Lys Tyr Gly Val Glu Thr Asp Trp Pro 355 360 365 Ala Tyr Ile Glu GlyTrp Lys Lys Leu Ala Thr Asp Glu Leu Glu Lys 370 375 380 Tyr Ala Lys AsnGlu Pro Thr Leu Ile Arg Ile Trp Gly Asp Ala Leu 385 390 395 400 Phe AspIle Val Asp Lys Asp Gln Asn Gly Ala Ile Thr Leu Asp Glu 405 410 415 TrpLys Ala Tyr Thr Lys Ala Ala Gly Ile Ile Gln Ser Ser Glu Asp 420 425 430Cys Glu Glu Thr Phe Arg Val Cys Asp Ile Asp Glu Ser Gly Gln Leu 435 440445 Asp Val Asp Glu Met Thr Arg Gln His Leu Gly Phe Trp Tyr Thr Met 450455 460 Asp Pro Ala Cys Glu Lys Leu Tyr Gly Gly Ala Val Pro 465 470 4756 906 PRT Aequorea victoria 6 Met Val Ser Ala Ser Arg Pro Glu Ala LeuAla Ala Pro Val Thr Thr 1 5 10 15 Val Ala Thr Leu Val Pro His Asn AlaThr Glu Pro Ala Ser Pro Gly 20 25 30 Glu Gly Lys Glu Asp Ala Phe Ser LysLeu Lys Gln Lys Phe Met Asn 35 40 45 Glu Leu His Lys Ile Pro Leu Pro ProTrp Ala Leu Ile Ala Ile Ala 50 55 60 Ile Val Ala Val Leu Leu Val Val ThrCys Cys Phe Cys Val Cys Lys 65 70 75 80 Lys Cys Leu Phe Lys Lys Lys AsnLys Lys Lys Gly Lys Glu Lys Gly 85 90 95 Gly Lys Asn Ala Ile Asn Met LysAsp Val Lys Asp Leu Gly Lys Thr 100 105 110 Met Lys Asp Gln Ala Leu LysAsp Asp Asp Ala Glu Thr Gly Leu Thr 115 120 125 Asp Gly Glu Glu Lys GluGlu Pro Lys Glu Glu Glu Lys Leu Gly Lys 130 135 140 Leu Gln Tyr Ser LeuAsp Tyr Asp Phe Gln Asn Asn Gln Leu Leu Val 145 150 155 160 Gly Ile IleGln Ala Ala Glu Leu Pro Ala Leu Asp Met Gly Gly Thr 165 170 175 Ser AspPro Tyr Val Lys Val Phe Leu Leu Pro Asp Lys Lys Lys Lys 180 185 190 PheGlu Thr Lys Val His Arg Lys Thr Leu Asn Pro Val Phe Asn Glu 195 200 205Gln Phe Thr Phe Lys Val Pro Tyr Ser Glu Leu Gly Gly Lys Thr Leu 210 215220 Val Met Ala Val Tyr Asp Phe Asp Arg Phe Ser Lys His Asp Ile Ile 225230 235 240 Gly Glu Phe Lys Val Pro Met Asn Thr Val Asp Phe Gly His ValThr 245 250 255 Glu Glu Trp Arg Asp Leu Gln Ser Ala Glu Lys Glu Glu GlnGlu Lys 260 265 270 Leu Gly Asp Ile Cys Phe Ser Leu Arg Tyr Val Pro ThrAla Gly Lys 275 280 285 Leu Thr Val Val Ile Leu Glu Ala Lys Asn Leu LysLys Met Asp Val 290 295 300 Gly Gly Leu Ser Asp Pro Tyr Val Lys Ile HisLeu Met Gln Asn Gly 305 310 315 320 Lys Arg Leu Lys Lys Lys Lys Thr ThrIle Lys Lys Asn Thr Leu Asn 325 330 335 Pro Tyr Tyr Asn Glu Ser Phe SerPhe Glu Val Pro Phe Glu Gln Ile 340 345 350 Gln Lys Val Gln Val Val ValThr Val Leu Asp Tyr Asp Lys Ile Gly 355 360 365 Lys Asn Asp Ala Ile GlyLys Val Phe Val Gly Tyr Asn Ser Thr Gly 370 375 380 Ala Glu Leu Arg HisTrp Ser Asp Met Leu Ala Asn Pro Arg Arg Pro 385 390 395 400 Ile Ala GlnTrp His Thr Leu Gln Val Glu Glu Glu Val Asp Ala Met 405 410 415 Leu AlaVal Lys Arg Ser Gly Asn Ser Gly Arg Ala Thr Met Ser Lys 420 425 430 GlyGlu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp 435 440 445Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly 450 455460 Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly 465470 475 480 Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr TyrGly 485 490 495 Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln HisAsp Phe 500 505 510 Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu ArgThr Ile Phe 515 520 525 Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala GluVal Lys Phe Glu 530 535 540 Gly Asp Thr Leu Val Asn Arg Ile Glu Leu LysGly Ile Asp Phe Lys 545 550 555 560 Glu Asp Gly Asn Ile Leu Gly His LysLeu Glu Tyr Asn Tyr Asn Ser 565 570 575 His Asn Val Tyr Ile Met Ala AspLys Gln Lys Asn Gly Ile Lys Ala 580 585 590 Asn Phe Lys Ile Arg His AsnIle Glu Asp Gly Ser Val Gln Leu Ala 595 600 605 Asp His Tyr Gln Gln AsnThr Pro Ile Gly Asp Gly Pro Val Leu Leu 610 615 620 Pro Asp Asn His TyrLeu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro 625 630 635 640 Asn Glu LysArg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala 645 650 655 Gly IleThr His Gly Met Asp Glu Leu Tyr Lys Ser Gly Gly Ser Gly 660 665 670 SerGly Gly Gln Ser Gly Gly Ser Gly Ser Gly Gly Gln Ser Gly Gly 675 680 685Ser Gly Ser Gly Gly Gln Ser Gly Gly Ser Gly Ser Gly Gly Gln Ser 690 695700 Gly Gly Ser Gly Ser Gly Gly Gln Ser Gly Leu Arg Ser Val Lys Leu 705710 715 720 Thr Ser Asp Phe Asp Asn Pro Arg Trp Ile Gly Arg His Lys HisMet 725 730 735 Phe Asn Phe Leu Asp Val Asn His Asn Gly Lys Ile Ser LeuAsp Glu 740 745 750 Met Val Tyr Lys Ala Ser Asp Ile Val Ile Asn Asn LeuGly Ala Thr 755 760 765 Pro Glu Gln Ala Lys Arg His Lys Asp Ala Val GluAla Phe Phe Gly 770 775 780 Gly Ala Gly Met Lys Tyr Gly Val Glu Thr AspTrp Pro Ala Tyr Ile 785 790 795 800 Glu Gly Trp Lys Lys Leu Ala Thr AspGlu Leu Glu Lys Tyr Ala Lys 805 810 815 Asn Glu Pro Thr Leu Ile Arg IleTrp Gly Asp Ala Leu Phe Asp Ile 820 825 830 Val Asp Lys Asp Gln Asn GlyAla Ile Thr Leu Asp Glu Trp Lys Ala 835 840 845 Tyr Thr Lys Ala Ala GlyIle Ile Gln Ser Ser Glu Asp Cys Glu Glu 850 855 860 Thr Phe Arg Val CysAsp Ile Asp Glu Ser Gly Gln Leu Asp Val Asp 865 870 875 880 Glu Met ThrArg Gln His Leu Gly Phe Trp Tyr Thr Met Asp Pro Ala 885 890 895 Cys GluLys Leu Tyr Gly Gly Ala Val Pro 900 905 7 3973 DNA Aequorea victoria 7atgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 60gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 120aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 180gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag 240cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 300aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 360aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 420ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc 480atcaaggcca acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac 540cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 600ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg 660ctggagttcg tgaccgccgc cgggatcact cacggcatgg acgagctgta caagtccgga 720ctcagatctg tcaaacttac atcagacttc gacaacccaa gatggattgg acgacacaag 780catatgttca atttccttga tgtcaaccac aatggaaaaa tctctcttga cgagatggtc 840tacaaggcat ctgatattgt catcaataac cttggagcaa cacctgagca agccaaacga 900cacaaagatg ctgtggaagc cttcttcgga ggagctggaa tgaaatatgg tgtggaaact 960gattggcctg catatattga aggatggaaa aaattggcta ctgatgaatt ggagaaatac 1020gccaaaaacg aaccaaccct catccgcata tggggtgatg ctttgtttga tatcgttgac 1080aaagatcaaa atggagctat tacactggat gaatggaaag catacaccaa agctgctggt 1140atcatccaat catcagaaga ttgcgaggaa acattcagag tgtgcgatat tgatgaaagt 1200ggacaactcg atgttgatga gatgacaaga cagcatctgg gattttggta caccatggat 1260cctgcttgcg aaaagctcta cggtggagct gtccccgaat gagcaagggc gaggagctgt 1320tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca 1380gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct 1440gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg 1500tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca 1560tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga 1620cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca 1680tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc 1740acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggccaac ttcaagatcc 1800gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca 1860tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga 1920gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg 1980ggatcactca cggcatggac gagctgtaca agtccggcgg gagcggatcc ggcggccagt 2040ccggcctcag atctgtcaaa cttacatcag acttcgacaa cccaagatgg attggacgac 2100acaagcatat gttcaatttc cttgatgtca accacaatgg aaaaatctct cttgacgaga 2160tggtctacaa ggcatctgat attgtcatca ataaccttgg agcaacacct gagcaagcca 2220aacgacacaa agatgctgtg gaagccttct tcggaggagc tggaatgaaa tatggtgtgg 2280aaactgattg gcctgcatat attgaaggat ggaaaaaatt ggctactgat gaattggaga 2340aatacgccaa aaacgaacca accctcatcc gcatatgggg tgatgctttg tttgatatcg 2400ttgacaaaga tcaaaatgga gctattacac tggatgaatg gaaagcatac accaaagctg 2460ctggtatcat ccaatcatca gaagattgcg aggaaacatt cagagtgtgc gatattgatg 2520aaagtggaca actcgatgtt gatgagatga caagacagca tctgggattt tggtacacca 2580tggatcctgc ttgcgaaaag ctctacggtg gagctgtccc cgaatgagca agggcgagga 2640gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa 2700gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt 2760catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta 2820cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc 2880cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta 2940caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa 3000gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa 3060cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg ccaacttcaa 3120gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac 3180ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc 3240cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc 3300cgccgggatc actcacggca tggacgagct gtacaagtcc ggcgggagcg gatccggcgg 3360ccagtccggc gggagcggat ccggcggcca gtccggcctc agatctgtca aacttacatc 3420agacttcgac aacccaagat ggattggacg acacaagcat atgttcaatt tccttgatgt 3480caaccacaat ggaaaaatct ctcttgacga gatggtctac aaggcatctg atattgtcat 3540caataacctt ggagcaacac ctgagcaagc caaacgacac aaagatgctg tggaagcctt 3600cttcggagga gctggaatga aatatggtgt ggaaactgat tggcctgcat atattgaagg 3660atggaaaaaa ttggctactg atgaattgga gaaatacgcc aaaaacgaac caaccctcat 3720ccgcatatgg ggtgatgctt tgtttgatat cgttgacaaa gatcaaaatg gagctattac 3780actggatgaa tggaaagcat acaccaaagc tgctggtatc atccaatcat cagaagattg 3840cgaggaaaca ttcagagtgt gcgatattga tgaaagtgga caactcgatg ttgatgagat 3900gacaagacag catctgggat tttggtacac catggatcct gcttgcgaaa agctctacgg 3960tggagctgtc ccc 3973 8 2673 DNA Aequorea victoria 8 atgagcaagg gcgaggagctgttcaccggg gtggtgccca tcctggtcga gctggacggc 60 gacgtaaacg gccacaagttcagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 120 aagctgaccc tgaagttcatctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 180 gtgaccaccc tgacctacggcgtgcagtgc ttcagccgct accccgacca catgaagcag 240 cacgacttct tcaagtccgccatgcccgaa ggctacgtcc aggagcgcac catcttcttc 300 aaggacgacg gcaactacaagacccgcgcc gaggtgaagt tcgagggcga caccctggtg 360 aaccgcatcg agctgaagggcatcgacttc aaggaggacg gcaacatcct ggggcacaag 420 ctggagtaca actacaacagccacaacgtc tatatcatgg ccgacaagca gaagaacggc 480 atcaaggcca acttcaagatccgccacaac atcgaggacg gcagcgtgca gctcgccgac 540 cactaccagc agaacacccccatcggcgac ggccccgtgc tgctgcccga caaccactac 600 ctgagcaccc agtccgccctgagcaaagac cccaacgaga agcgcgatca catggtcctg 660 ctggagttcg tgaccgccgccgggatcact cacggcatgg acgagctgta caagtccggc 720 gggagcggat ccggcggccagtccggcctc agatctgtca aacttacatc agacttcgac 780 aacccaagat ggattggacgacacaagcat atgttcaatt tccttgatgt caaccacaat 840 ggaaaaatct ctcttgacgagatggtctac aaggcatctg atattgtcat caataacctt 900 ggagcaacac ctgagcaagccaaacgacac aaagatgctg tggaagcctt cttcggagga 960 gctggaatga aatatggtgtggaaactgat tggcctgcat atattgaagg atggaaaaaa 1020 ttggctactg atgaattggagaaatacgcc aaaaacgaac caaccctcat ccgcatatgg 1080 ggtgatgctt tgtttgatatcgttgacaaa gatcaaaatg gagctattac actggatgaa 1140 tggaaagcat acaccaaagctgctggtatc atccaatcat cagaagattg cgaggaaaca 1200 ttcagagtgt gcgatattgatgaaagtgga caactcgatg ttgatgagat gacaagacag 1260 catctgggat tttggtacaccatggatcct gcttgcgaaa agctctacgg tggagctgtc 1320 cccatgagca agggcgaggagctgttcacc ggggtggtgc ccatcctggt cgagctggac 1380 ggcgacgtaa acggccacaagttcagcgtg tccggcgagg gcgagggcga tgccacctac 1440 ggcaagctga ccctgaagttcatctgcacc accggcaagc tgcccgtgcc ctggcccacc 1500 ctcgtgacca ccctgacctacggcgtgcag tgcttcagcc gctaccccga ccacatgaag 1560 cagcacgact tcttcaagtccgccatgccc gaaggctacg tccaggagcg caccatcttc 1620 ttcaaggacg acggcaactacaagacccgc gccgaggtga agttcgaggg cgacaccctg 1680 gtgaaccgca tcgagctgaagggcatcgac ttcaaggagg acggcaacat cctggggcac 1740 aagctggagt acaactacaacagccacaac gtctatatca tggccgacaa gcagaagaac 1800 ggcatcaagg ccaacttcaagatccgccac aacatcgagg acggcagcgt gcagctcgcc 1860 gaccactacc agcagaacacccccatcggc gacggccccg tgctgctgcc cgacaaccac 1920 tacctgagca cccagtccgccctgagcaaa gaccccaacg agaagcgcga tcacatggtc 1980 ctgctggagt tcgtgaccgccgccgggatc actcacggca tggacgagct gtacaagtcc 2040 ggcgggagcg gatccggcggccagtccggc gggagcggat ccggcggcca gtccggcctc 2100 agatctgtca aacttacatcagacttcgac aacccaagat ggattggacg acacaagcat 2160 atgttcaatt tccttgatgtcaaccacaat ggaaaaatct ctcttgacga gatggtctac 2220 aaggcatctg atattgtcatcaataacctt ggagcaacac ctgagcaagc caaacgacac 2280 aaagatgctg tggaagccttcttcggagga gctggaatga aatatggtgt ggaaactgat 2340 tggcctgcat atattgaaggatggaaaaaa ttggctactg atgaattgga gaaatacgcc 2400 aaaaacgaac caaccctcatccgcatatgg ggtgatgctt tgtttgatat cgttgacaaa 2460 gatcaaaatg gagctattacactggatgaa tggaaagcat acaccaaagc tgctggtatc 2520 atccaatcat cagaagattgcgaggaaaca ttcagagtgt gcgatattga tgaaagtgga 2580 caactcgatg ttgatgagatgacaagacag catctgggat tttggtacac catggatcct 2640 gcttgcgaaa agctctacggtggagctgtc ccc 2673 9 1350 DNA Aequorea victoria 9 atgagcaagg gcgaggagctgttcaccggg gtggtgccca tcctggtcga gctggacggc 60 gacgtaaacg gccacaagttcagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 120 aagctgaccc tgaagttcatctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 180 gtgaccaccc tgacctacggcgtgcagtgc ttcagccgct accccgacca catgaagcag 240 cacgacttct tcaagtccgccatgcccgaa ggctacgtcc aggagcgcac catcttcttc 300 aaggacgacg gcaactacaagacccgcgcc gaggtgaagt tcgagggcga caccctggtg 360 aaccgcatcg agctgaagggcatcgacttc aaggaggacg gcaacatcct ggggcacaag 420 ctggagtaca actacaacagccacaacgtc tatatcatgg ccgacaagca gaagaacggc 480 atcaaggcca acttcaagatccgccacaac atcgaggacg gcagcgtgca gctcgccgac 540 cactaccagc agaacacccccatcggcgac ggccccgtgc tgctgcccga caaccactac 600 ctgagcaccc agtccgccctgagcaaagac cccaacgaga agcgcgatca catggtcctg 660 ctggagttcg tgaccgccgccgggatcact cacggcatgg acgagctgta caagtccggc 720 gggagcggat ccggcggccagtccggcggg agcggatccg gcggccagtc cggcctcaga 780 tctgtcaaac ttacatcagacttcgacaac ccaagatgga ttggacgaca caagcatatg 840 ttcaatttcc ttgatgtcaaccacaatgga aaaatctctc ttgacgagat ggtctacaag 900 gcatctgata ttgtcatcaataaccttgga gcaacacctg agcaagccaa acgacacaaa 960 gatgctgtgg aagccttcttcggaggagct ggaatgaaat atggtgtgga aactgattgg 1020 cctgcatata ttgaaggatggaaaaaattg gctactgatg aattggagaa atacgccaaa 1080 aacgaaccaa ccctcatccgcatatggggt gatgctttgt ttgatatcgt tgacaaagat 1140 caaaatggag ctattacactggatgaatgg aaagcataca ccaaagctgc tggtatcatc 1200 caatcatcag aagattgcgaggaaacattc agagtgtgcg atattgatga aagtggacaa 1260 ctcgatgttg atgagatgacaagacagcat ctgggatttt ggtacaccat ggatcctgct 1320 tgcgaaaagc tctacggtggagctgtcccc 1350 10 1404 DNA Aequorea victoria 10 atgagcaagg gcgaggagctgttcaccggg gtggtgccca tcctggtcga gctggacggc 60 gacgtaaacg gccacaagttcagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 120 aagctgaccc tgaagttcatctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 180 gtgaccaccc tgacctacggcgtgcagtgc ttcagccgct accccgacca catgaagcag 240 cacgacttct tcaagtccgccatgcccgaa ggctacgtcc aggagcgcac catcttcttc 300 aaggacgacg gcaactacaagacccgcgcc gaggtgaagt tcgagggcga caccctggtg 360 aaccgcatcg agctgaagggcatcgacttc aaggaggacg gcaacatcct ggggcacaag 420 ctggagtaca actacaacagccacaacgtc tatatcatgg ccgacaagca gaagaacggc 480 atcaaggcca acttcaagatccgccacaac atcgaggacg gcagcgtgca gctcgccgac 540 cactaccagc agaacacccccatcggcgac ggccccgtgc tgctgcccga caaccactac 600 ctgagcaccc agtccgccctgagcaaagac cccaacgaga agcgcgatca catggtcctg 660 ctggagttcg tgaccgccgccgggatcact cacggcatgg acgagctgta caagtccggc 720 gggagcggat ccggcggccagtccggcggg agcggatccg gcggccagtc cggcgggagc 780 ggatccggcg gccagtccggcgggagcgga tccggcggcc agtccggcct cagatctgtc 840 aaacttacat cagacttcgacaacccaaga tggattggac gacacaagca tatgttcaat 900 ttccttgatg tcaaccacaatggaaaaatc tctcttgacg agatggtcta caaggcatct 960 gatattgtca tcaataaccttggagcaaca cctgagcaag ccaaacgaca caaagatgct 1020 gtggaagcct tcttcggaggagctggaatg aaatatggtg tggaaactga ttggcctgca 1080 tatattgaag gatggaaaaaattggctact gatgaattgg agaaatacgc caaaaacgaa 1140 ccaaccctca tccgcatatggggtgatgct ttgtttgata tcgttgacaa agatcaaaat 1200 ggagctatta cactggatgaatggaaagca tacaccaaag ctgctggtat catccaatca 1260 tcagaagatt gcgaggaaacattcagagtg tgcgatattg atgaaagtgg acaactcgat 1320 gttgatgaga tgacaagacagcatctggga ttttggtaca ccatggatcc tgcttgcgaa 1380 aagctctacg gtggagctgtcccc 1404 11 1431 DNA Aequorea victoria 11 atgagcaagg gcgaggagctgttcaccggg gtggtgccca tcctggtcga gctggacggc 60 gacgtaaacg gccacaagttcagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 120 aagctgaccc tgaagttcatctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 180 gtgaccaccc tgacctacggcgtgcagtgc ttcagccgct accccgacca catgaagcag 240 cacgacttct tcaagtccgccatgcccgaa ggctacgtcc aggagcgcac catcttcttc 300 aaggacgacg gcaactacaagacccgcgcc gaggtgaagt tcgagggcga caccctggtg 360 aaccgcatcg agctgaagggcatcgacttc aaggaggacg gcaacatcct ggggcacaag 420 ctggagtaca actacaacagccacaacgtc tatatcatgg ccgacaagca gaagaacggc 480 atcaaggcca acttcaagatccgccacaac atcgaggacg gcagcgtgca gctcgccgac 540 cactaccagc agaacacccccatcggcgac ggccccgtgc tgctgcccga caaccactac 600 ctgagcaccc agtccgccctgagcaaagac cccaacgaga agcgcgatca catggtcctg 660 ctggagttcg tgaccgccgccgggatcact cacggcatgg acgagctgta caagtccggc 720 gggagcggat ccggcggccagtccggcggg agcggatccg gcggccagtc cggcgggagc 780 ggatccggcg gccagtccggcgggagcgga tccggcggcc agtccggcgg gagcggatcc 840 ggcggccagt ccggcctcagatctgtcaaa cttacatcag acttcgacaa cccaagatgg 900 attggacgac acaagcatatgttcaatttc cttgatgtca accacaatgg aaaaatctct 960 cttgacgaga tggtctacaaggcatctgat attgtcatca ataaccttgg agcaacacct 1020 gagcaagcca aacgacacaaagatgctgtg gaagccttct tcggaggagc tggaatgaaa 1080 tatggtgtgg aaactgattggcctgcatat attgaaggat ggaaaaaatt ggctactgat 1140 gaattggaga aatacgccaaaaacgaacca accctcatcc gcatatgggg tgatgctttg 1200 tttgatatcg ttgacaaagatcaaaatgga gctattacac tggatgaatg gaaagcatac 1260 accaaagctg ctggtatcatccaatcatca gaagattgcg aggaaacatt cagagtgtgc 1320 gatattgatg aaagtggacaactcgatgtt gatgagatga caagacagca tctgggattt 1380 tggtacacca tggatcctgcttgcgaaaag ctctacggtg gagctgtccc c 1431 12 2718 DNA Aequorea victoria 12atggtgagtg ccagtcgtcc tgaggccctg gctgcccctg tcaccactgt tgcgaccctt 60gtcccacaca acgccactga gccagccagt cctggggaag ggaaggaaga tgccttttcc 120aagctgaagc agaagtttat gaatgaactg cataaaatcc cattgccacc gtgggcctta 180attgccatag ccatagttgc ggtccttcta gtcgtgacct gctgcttctg tgtctgtaag 240aaatgtttgt tcaaaaagaa aaacaagaag aagggaaagg aaaagggagg gaagaacgcc 300attaacatga aagacgtgaa agacttaggg aagaccatga aggatcaggc ccttaaggat 360gacgatgctg aaactggact gactgatgga gaagaaaagg aggagcccaa ggaagaggag 420aaactgggaa agcttcaata ttcactggac tatgacttcc agaataacca gctgctggtg 480ggaatcatcc aggctgctga actgcccgcc ctggacatgg gaggcacatc tgatccatac 540gtcaaagtct tcctgctgcc cgacaaaaag aagaagtttg agacaaaagt ccaccggaaa 600accctcaatc cagtcttcaa tgaacagttt actttcaagg tgccatactc ggaattaggt 660ggcaagacac tggtgatggc tgtgtatgat tttgaccgct tctccaagca cgacatcatt 720ggagagttca aagttcctat gaacaccgtg gattttggcc acgtcaccga ggagtggcgc 780gatctccaga gtgctgagaa agaagagcaa gagaaactgg gtgacatctg cttctccctc 840cgctacgtcc ctactgccgg caagctgact gttgtcattc tggaagccaa gaacctgaag 900aagatggatg tgggtggctt atctgatccc tatgtaaaga ttcacctgat gcagaacggc 960aagagactga agaagaaaaa gacaacgatt aagaagaaca cacttaaccc ctactacaat 1020gagtccttca gctttgaagt tccgttcgag caaatccaga aagtgcaagt ggtggtaact 1080gttttggact atgacaagat tggcaagaac gacgccatcg gcaaagtctt tgtgggctac 1140aacagcaccg gcgcagagct gcgacactgg tcagacatgc tggccaaccc ccggcgaccc 1200atcgcccagt ggcacactct gcaggtagag gaggaggttg atgccatgct ggctgtcaag 1260agatccggga attccgggcg ggccaccatg agcaagggcg aggagctgtt caccggggtg 1320gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc 1380gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc 1440aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc 1500agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc 1560tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag 1620gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag 1680gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat 1740atcatggccg acaagcagaa gaacggcatc aaggccaact tcaagatccg ccacaacatc 1800gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc 1860cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc 1920aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactcac 1980ggcatggacg agctgtacaa gtccggcggg agcggatccg gcggccagtc cggcgggagc 2040ggatccggcg gccagtccgg cgggagcgga tccggcggcc agtccggcgg gagcggatcc 2100ggcggccagt ccggcgggag cggatccggc ggccagtccg gcctcagatc tgtcaaactt 2160acatcagact tcgacaaccc aagatggatt ggacgacaca agcatatgtt caatttcctt 2220gatgtcaacc acaatggaaa aatctctctt gacgagatgg tctacaaggc atctgatatt 2280gtcatcaata accttggagc aacacctgag caagccaaac gacacaaaga tgctgtggaa 2340gccttcttcg gaggagctgg aatgaaatat ggtgtggaaa ctgattggcc tgcatatatt 2400gaaggatgga aaaaattggc tactgatgaa ttggagaaat acgccaaaaa cgaaccaacc 2460ctcatccgca tatggggtga tgctttgttt gatatcgttg acaaagatca aaatggagct 2520attacactgg atgaatggaa agcatacacc aaagctgctg gtatcatcca atcatcagaa 2580gattgcgagg aaacattcag agtgtgcgat attgatgaaa gtggacaact cgatgttgat 2640gagatgacaa gacagcatct gggattttgg tacaccatgg atcctgcttg cgaaaagctc 2700tacggtggag ctgtcccc 2718 13 15 DNA Artificial Sequence Description ofArtificial Sequence DNA sequence of GFP-aequorin linker 13 tccggcctcagatct 15 14 42 DNA Artificial Sequence Description of ArtificialSequence DNA sequence of GFP-aequorin linker 14 tccggcggga gcggatccggcggccagtcc ggcctcagat ct 42 15 69 DNA Artificial Sequence Description ofArtificial Sequence DNA sequence of GFP-aequorin linker 15 tccggcgggagcggatccgg cggccagtcc ggcgggagcg gatccggcgg ccagtccggc 60 ctcagatct 6916 123 DNA Artificial Sequence Description of Artificial Sequence DNAsequence of GFP-aequorin linker 16 tccggcggga gcggatccgg cggccagtccggcgggagcg gatccggcgg ccagtccggc 60 gggagcggat ccggcggcca gtccggcgggagcggatccg gcggccagtc cggcctcaga 120 tct 123 17 150 DNA ArtificialSequence Description of Artificial Sequence DNA sequence of GFP-aequorinlinker 17 tccggcggga gcggatccgg cggccagtcc ggcgggagcg gatccggcggccagtccggc 60 gggagcggat ccggcggcca gtccggcggg agcggatccg gcggccagtccggcgggagc 120 ggatccggcg gccagtccgg cctcagatct 150 18 5 PRT ArtificialSequence Description of Artificial Sequence Peptide sequence of linker18 Ser Gly Leu Arg Ser 1 5 19 14 PRT Artificial Sequence Description ofArtificial Sequence Peptide sequence of linker 19 Ser Gly Gly Ser GlySer Gly Gly Gln Ser Gly Leu Arg Ser 1 5 10 20 23 PRT Artificial SequenceDescription of Artificial Sequence Peptide sequence of linker 20 Ser GlyGly Ser Gly Ser Gly Gly Gln Ser Gly Gly Ser Gly Ser Gly 1 5 10 15 GlyGln Ser Gly Leu Arg Ser 20 21 41 PRT Artificial Sequence Description ofArtificial Sequence Peptide sequence of linker 21 Ser Gly Gly Ser GlySer Gly Gly Gln Ser Gly Gly Ser Gly Ser Gly 1 5 10 15 Gly Gln Ser GlyGly Ser Gly Ser Gly Gly Gln Ser Gly Gly Ser Gly 20 25 30 Ser Gly Gly GlnSer Gly Leu Arg Ser 35 40 22 50 PRT Artificial Sequence Description ofArtificial Sequence Peptide sequence of linker 22 Ser Gly Gly Ser GlySer Gly Gly Gln Ser Gly Gly Ser Gly Ser Gly 1 5 10 15 Gly Gln Ser GlyGly Ser Gly Ser Gly Gly Gln Ser Gly Gly Ser Gly 20 25 30 Ser Gly Gly GlnSer Gly Gly Ser Gly Ser Gly Gly Gln Ser Gly Leu 35 40 45 Arg Ser 50 2327 DNA Artificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 23 ccggcgggag cggatccggc ggccagt 27 24 27 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 24ccggactggc cgccggatcc gctcccg 27 25 45 PRT Artificial SequenceDescription of Artificial Sequence Linker 25 Gly Gly Ser Gly Ser Gly GlyGln Ser Gly Gly Ser Gly Ser Gly Gly 1 5 10 15 Gln Ser Gly Gly Ser GlySer Gly Gly Gln Ser Gly Gly Ser Gly Ser 20 25 30 Gly Gly Gln Ser Gly GlySer Gly Ser Gly Gly Gln Ser 35 40 45 26 5 PRT Artificial SequenceDescription of Artificial Sequence Linker 26 Ser Gly Leu Arg Ser 1 5 2727 DNA Unknown Organism Description of Unknown Organism pEGFP-Cl plasmid27 gtcgacggta ccgcgggccc gggatcc 27 28 14 DNA Artificial SequenceDescription of Artificial Sequence Illustrative nucleic acid 28gtcgacgggg atcc 14 29 33 DNA Artificial Sequence Description ofArtificial Sequence Synthetic construct 29 gcgctaccgc gggccacc atg agcaag ggc gag 33 Met Ser Lys Gly Glu 1 5 30 5 PRT Artificial SequenceDescription of Artificial Sequence Synthetic construct 30 Met Ser LysGly Glu 1 5 31 36 DNA Aequorea victoria CDS (19)..(36) 31 gcgctaccggtcgccacc atg gtg agc aag ggc gag 36 Met Val Ser Lys Gly Glu 1 5 32 6 PRTAequorea victoria 32 Met Val Ser Lys Gly Glu 1 5 33 20 DNA ArtificialSequence Description of Artificial Sequence Synthetic construct 33 gcatc aag gcc aac ttc aag 20 Ile Lys Ala Asn Phe Lys 1 5 34 6 PRTArtificial Sequence Description of Artificial Sequence Syntheticconstruct 34 Ile Lys Ala Asn Phe Lys 1 5 35 20 DNA Aequorea victoria CDS(3)..(20) 35 gc atc aag gtg aac ttc aag 20 Ile Lys Val Asn Phe Lys 1 536 6 PRT Aequorea victoria 36 Ile Lys Val Asn Phe Lys 1 5 37 19 DNAArtificial Sequence Description of Artificial Sequence Syntheticconstruct 37 gg atc act cac ggc atg ga 19 Ile Thr His Gly Met 1 5 38 5PRT Artificial Sequence Description of Artificial Sequence Syntheticconstruct 38 Ile Thr His Gly Met 1 5 39 19 DNA Aequorea victoria CDS(3)..(17) 39 gg atc act ctc ggc atg ga 19 Ile Thr Leu Gly Met 1 5 40 5PRT Aequorea victoria 40 Ile Thr Leu Gly Met 1 5 41 596 DNA ArtificialSequence Description of Artificial Sequence Altered Aequoria victoriasequence 41 agcttcagat ctgtcaaact tacatcagac ttcgacaacc caagatggattggacgacac 60 aagcatatgt tcaatttcct tgatgtcaac cacaatggaa aaatctctcttgacgagatg 120 gtctacaagg catctgatat tgtcatcaat aaccttggag caacacctgagcaagccaaa 180 cgacacaaag atgctgtgga agccttcttc ggaggagctg gaatgaaatatggtgtggaa 240 actgattggc ctgcatatat tgaaggatgg aaaaaattgg ctactgatgaattggagaaa 300 tacgccaaaa acgaaccaac cctcatccgc atctggggtg atgctttgtttgatatcgtt 360 gacaaagatc aaaatggagc tattacactg gatgaatgga aagcatacaccaaagctgct 420 ggtatcatcc aatcatcaga agattgcgag gaaacattca gagtgtgcgatattgatgaa 480 agtggacaac tcgatgttga tgagatgaca agacagcatc tgggattttggtacaccatg 540 gatcctgctt gcgaaaagct ctacggtgga gctgtcccct aatctcgaggatcttt 596 42 21 DNA Artificial Sequence Description of ArtificialSequence Synthetic construct 42 aag tcc gga ctc aga tct gtc 21 Lys SerGly Leu Arg Ser Val 1 5 43 7 PRT Artificial Sequence Description ofArtificial Sequence Synthetic construct 43 Lys Ser Gly Leu Arg Ser Val 15 44 21 DNA Artificial Sequence Description of Artificial SequenceSynthetic construct 44 gacagatctg agtccggact t 21 45 21 DNA ArtificialSequence Description of Artificial Sequence Synthetic construct 45aagtgcggac tcagatctgt c 21 46 27 DNA Artificial Sequence Description ofArtificial Sequence Synthetic construct 46 ccggcgggag cggatccggc ggccagt27 47 9 PRT Artificial Sequence Description of Artificial SequenceSynthetic construct 47 Gly Gly Ser Gly Ser Gly Gly Gln Ser 1 5 48 27 DNAArtificial Sequence Description of Artificial Sequence Syntheticconstruct 48 ccggactggc cgccggatcc gctcccg 27

What is claimed is:
 1. A purified polypeptide having the amino acidsequence of SEQ ID NO:
 5. 2. A peptide linker comprising the amino acidsequence of SEQ ID NO:
 22. 3. A fusion protein comprising twobioluminescent proteins and a peptide linker according to claim
 2. 4.The fusion protein according to claim 3, wherein said two bioluminescentproteins comprise at feast an aequorin protein.
 5. The fusion protein ofclaim 3 comprising aequorin protein and a GFP protein.
 6. A fusionprotein of the formula: GFP-LINKER-AEQ: wherein GFP is green fluorescentprotein; AEQ is aequorin; and LINKER is a polypeptide of 14-50 aminoacids.
 7. A composition comprising the fusion protein of claim 6,wherein the composition binds calcium ions and transmits measurableenergy, wherein the amount of energy depends on the quantity of calciumbound and the quantity of peptide in the composition in absence of anylight excitation.
 8. The composition according to claim 7, wherein thefusion protein comprises SEQ ID NO:
 5. 9. A kit for measuring thetransfer of energy in vivo or in vitro comprising the fusion protein ofclaim 6 and reagents necessary for visualizing or detecting transfer ofenergy in the presence or in the absence of a molecule of interest. 10.A method of screening in vitro a change in a physical, chemical,biochemical, or biological condition, wherein the method comprises: (a)adding into a reaction system a composition according to claim 7containing an analyte of interest in presence or in absence of amolecule of interest to be tested; and (b) visualizing the emission ofenergy produced in step (a).
 11. A method of screening of a productleading to a change in a physical, chemical, biochemical or biologicalcondition, wherein the method comprises: (a) contacting a biologicalsample from a vertebrate with a pharmaceutically acceptable mediumcomprising the composition according to claim 7 in presence or inabsence of a molecule of interest to be tested; (b) detecting energyproduced in presence of said composition; and (c) optionally, measuringthe effective concentration of said molecule of interest necessary forthe detection of the energy in step (b).
 12. A method of screening invitro for a molecule in a biological sample that inhibits or increasesthe measurable energy in the composition of claim 7, wherein themolecule is contained in a reaction system, wherein the methodcomprises: (a) detecting an increase or decrease of the energy in thereaction system by comparison with a control reaction system containingthe composition of claim 7 without the molecule to be tested; and (b)optionally, determining the effective minimal concentration of themolecule capable of inhibiting or increasing the energy transfer of thecomposition in the reaction system.